( H ) TIRF images ( upper ) and TIRF - Z scan ( lower ) of the same resting and store depleted cells HEK cells transfected with ANO8 - YFP alone . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . The experiments represent at least 4 separate experiments with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) TIRF images (upper) and TIRF-Z scan (lower) of the same resting and store depleted cells HEK cells transfected with ANO8-YFP alone. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. The experiments represent at least 4 separate experiments with similar results.


( I ) : Confocal images of resting cells transfected with ANO8 - YFP , STIM1 - CFP and Orai1 - mCherry . ( J ) Confocal images of store depleted cells transfected with ANO8 - YFP , STIM1 - CFP and Orai1 - mCherry . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . The experiments represent at least 4 separate experiments with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I): Confocal images of resting cells transfected with ANO8-YFP, STIM1-CFP and Orai1-mCherry. (J) Confocal images of store depleted cells transfected with ANO8-YFP, STIM1-CFP and Orai1-mCherry. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. The experiments represent at least 4 separate experiments with similar results.


( K ) TIRF - Z scan of resting ( upper ) and store depleted ( lower ) cells transfected with ANO8 - YFP and STIM1 - mCherry demonstrating the relationship between STIM1 and ANO8 puncta at the TIRF field . Cell surface is on top . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . The experiments represent at least 4 separate experiments with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) TIRF-Z scan of resting (upper) and store depleted (lower) cells transfected with ANO8-YFP and STIM1-mCherry demonstrating the relationship between STIM1 and ANO8 puncta at the TIRF field. Cell surface is on top. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. The experiments represent at least 4 separate experiments with similar results.


( A ) FRET ratio was measured with HEK cells transfected with STIM1 - CFP , Orai1 - YFP ( black ) and co - transfected with untagged ANO8 ( red ) before and after store depletion with 25 µM CPA in Ca2 + - free solution . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) FRET ratio was measured with HEK cells transfected with STIM1-CFP, Orai1-YFP (black) and co-transfected with untagged ANO8 (red) before and after store depletion with 25 µM CPA in Ca2+-free solution. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


TIRF microscopy was used to quantify puncta formation at the TIRF field in cells transfected with STIM1 - CFP , ANO8 - YFP and Orai1 - mCherry before and after store depletion . ( A ) Example images . ( B ) Time course of STIM1 puncta formation in cells transfected with STIM1 and Orai1 alone ( black ) or together with ANO8 ( red ) . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

TIRF microscopy was used to quantify puncta formation at the TIRF field in cells transfected with STIM1-CFP, ANO8-YFP and Orai1-mCherry before and after store depletion. (A) Example images. (B) Time course of STIM1 puncta formation in cells transfected with STIM1 and Orai1 alone (black) or together with ANO8 (red). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( C - E ) Effect of ANO8 on the rate of STIM1 - STIM1 clustering ( C ) , number of STIM1 puncta ( D ) and number of Orai1 puncta ( E ) . The results are mean ± s . e . m and difference were analyzed by unpaired t test . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-E) Effect of ANO8 on the rate of STIM1-STIM1 clustering (C), number of STIM1 puncta (D) and number of Orai1 puncta (E). The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( F ) HEK cells were used to immunoprecipitate ( IP ) the native ANO8 ( A8 ) or Orai1 ( O1 ) and blotted ( B ) for STIM1 . R = resting , S = stimulated . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) HEK cells were used to immunoprecipitate (IP) the native ANO8 (A8) or Orai1 (O1) and blotted (B) for STIM1. R=resting, S=stimulated. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( G ) HEK cells treated with scrambled ( siScr ) or siANO8 ( siA8 ) were used to IP the native Orai1 ( O1 ) or STIM1 ( S1 ) and blotted ( B ) for the native STIM1 , Orai1 or ANO8 , as indicated . The columns show the mean ± s . e . m of 4 independent experiments . The results are mean ± s . e . m and difference were analyzed by unpaired t test . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) HEK cells treated with scrambled (siScr) or siANO8 (siA8) were used to IP the native Orai1 (O1) or STIM1 (S1) and blotted (B) for the native STIM1, Orai1 or ANO8, as indicated. The columns show the mean±s.e.m of 4 independent experiments. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( H ) Co - IP of myc - STIM1 and Orai1 - HA , myc - STIM1 and ANO8 - YFP , and Orai1 - HA and ANO8 - YFP was measured in transfected HEK cells before ( R , resting ) and in response to store depletion ( S , stimulated ) . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Co-IP of myc-STIM1 and Orai1-HA, myc-STIM1 and ANO8-YFP, and Orai1-HA and ANO8-YFP was measured in transfected HEK cells before (R, resting) and in response to store depletion (S, stimulated). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( I ) Effect of ANO8 - YFP on myc - STIM1 and Orai1 - HA at the ER / PM junction . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Effect of ANO8-YFP on myc-STIM1 and Orai1-HA at the ER/PM junction. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( J - L ) Junctional Orai1 ( J ) , STIM1 ( K ) and ANO8 ( L ) in resting ( R ) and store - depleted cells ( S ) . The results are mean ± s . e . m of 4 experiments and difference were analyzed by unpaired t test . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J-L) Junctional Orai1 (J), STIM1 (K) and ANO8 (L) in resting (R) and store-depleted cells (S). The results are mean±s.e.m of 4 experiments and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( M ) Example EM images recorded from HEK cells transfected with empty vector ( left ) or ANO8 ( right ) . Red arrows mark ER / PM junctions . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) Example EM images recorded from HEK cells transfected with empty vector (left) or ANO8 (right). Red arrows mark ER/PM junctions. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( N , O ) The size ( N ) and density ( O ) of ER / PM junctions in vector - transfected ( control ) and ANO8 - transfected cells . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(N, O) The size (N) and density (O) of ER/PM junctions in vector-transfected (control) and ANO8-transfected cells. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( A , B ) Knockdown of ANO8 ( siA8 ) reduced CRAC current in cells transfected with Orai1 ( O1 ) and STIM1 ( S1 ) and buffered with 3 mM EGTA . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Knockdown of ANO8 (siA8) reduced CRAC current in cells transfected with Orai1 (O1) and STIM1 (S1) and buffered with 3 mM EGTA. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( C ) Knockdown of ANO8 reduces the native store - dependent Ca2 + influx measured in store depleted cells by Ca2 + add - back . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test .

O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Knockdown of ANO8 reduces the native store-dependent Ca2+ influx measured in store depleted cells by Ca2+ add-back. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( D , E ) Knockdown of ANO8 reduced the number of store - dependent STIM1 puncta at the TIRF plane in cells expressing STIM1 and Orai1 . Panel ( D ) shows representative images and ( E ) is the summary of 7 experiments . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test .

O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D,E) Knockdown of ANO8 reduced the number of store-dependent STIM1 puncta at the TIRF plane in cells expressing STIM1 and Orai1. Panel (D) shows representative images and (E) is the summary of 7 experiments. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( F , G ) Current was measured with pipette solution contacting the fast and strong Ca2 + buffer 10 mM BAPTA in HEK cells transfected with STIM1 , Orai1 , and with ( red ) or without ANO8 ( black ) . Panel ( G ) shows the increase in current density at peak current . Note the prominent current inactivation in the presence of ANO8 . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F, G) Current was measured with pipette solution contacting the fast and strong Ca2+ buffer 10 mM BAPTA in HEK cells transfected with STIM1, Orai1, and with (red) or without ANO8 (black). Panel (G) shows the increase in current density at peak current. Note the prominent current inactivation in the presence of ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( H ) Knockdown of SARAF ( red ) in wild - type cells had no effect on current inactivation in the presence of 10 mM BAPTA . ( I ) Knockdown of SARAF did not prevent the ANO8 - dependent current inactivation in the presence of 10 mM BAPTA . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Knockdown of SARAF (red) in wild-type cells had no effect on current inactivation in the presence of 10 mM BAPTA. (I) Knockdown of SARAF did not prevent the ANO8-dependent current inactivation in the presence of 10 mM BAPTA. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( A , B ) Depletion of plasma membrane PI ( 4 , 5 ) P2 with the FRB / FKBP system prevented the ANO8 - mediated increase in STIM1 - Orai1 current measured in pipette solutions containing 3 mM EGTA ( A ) or 10 mM BAPTA ( B ) . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Depletion of plasma membrane PI(4,5)P2 with the FRB/FKBP system prevented the ANO8-mediated increase in STIM1-Orai1 current measured in pipette solutions containing 3 mM EGTA (A) or 10 mM BAPTA (B). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( C ) HEK cells transfected with ANO8 - YFP , STIM1 - CFP Orai1 - HA and FRB and mCherry - FKBP PI ( 4 , 5 ) P2 depleting constructs were treated with 0 . 2 µM rapamycin ( rapa ) for 5 min and then with 25 µM CPA for 10 min before fixation and imaging by confocal microscopy . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) HEK cells transfected with ANO8-YFP, STIM1-CFP Orai1-HA and FRB and mCherry-FKBP PI(4,5)P2 depleting constructs were treated with 0.2 µM rapamycin (rapa) for 5 min and then with 25 µM CPA for 10 min before fixation and imaging by confocal microscopy. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( D ) Mutating ANO8 R948 , R950 , R951 in RPRRP , a region that is predicted to include an ANO8 PI ( 4 , 5 ) P2 binding site , prevented increased STIM1 - Orai1 current and inactivation . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Mutating ANO8 R948, R950, R951 in RPRRP, a region that is predicted to include an ANO8 PI(4,5)P2 binding site, prevented increased STIM1-Orai1 current and inactivation. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( E ) Mutating ANO8 R949 , 950 , 951 to Q reduced translocation of ANO8 . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Mutating ANO8 R949,950,951 to Q reduced translocation of ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( A ) Plot of the slope of inactivation as a function of current density in the presence ( red ) and absence of ANO8 ( black ) . Increasing current density was obtained by varying external Ca2 + between 2 - 50 mM . The results are mean ± s . e . m and difference were analyzed by unpaired t test . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Plot of the slope of inactivation as a function of current density in the presence (red) and absence of ANO8 (black). Increasing current density was obtained by varying external Ca2+ between 2-50 mM. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( B , C ) CRAC current was measured in HEK cells expressing STIM1 and Orai1 and in pipette solution containing 3 mM EGTA ( b ) or 10 mM BAPTA ( c ) . SERCA pump was inhibited with 25 µM CPA in the red traces where indicated and 100 µM IP3 was included in the pipette solution ( green in c ) . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, C) CRAC current was measured in HEK cells expressing STIM1 and Orai1 and in pipette solution containing 3 mM EGTA (b) or 10 mM BAPTA (c). SERCA pump was inhibited with 25 µM CPA in the red traces where indicated and 100 µM IP3 was included in the pipette solution (green in c). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( D , E ) Experimental protocol and conditions except that cells were also transfected with ANO8 . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Experimental protocol and conditions except that cells were also transfected with ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( F , G ) HEK cells expressing STIM1 , Orai1 , and ANO8 were used to measure current in pipette solution containing 10 mM BAPTA and with ( red ) or without ( black ) 100 µM IP3 or 10 mM of the ER Ca2 + chelator TPEN ( green ) . The current density ( F ) and the normalized current ( G ) are shown , illustrating the delay in current inactivation by IP3 and TPEN . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F, G) HEK cells expressing STIM1, Orai1, and ANO8 were used to measure current in pipette solution containing 10 mM BAPTA and with (red) or without (black) 100 µM IP3 or 10 mM of the ER Ca2+ chelator TPEN (green). The current density (F) and the normalized current (G) are shown, illustrating the delay in current inactivation by IP3 and TPEN. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( A , B ) : ER Ca2 + content was measured with ER - GECO1 in the periphery ( A ) and the center ( B ) or cells transfected with M3 receptors and with ( red , green ) or without ( black , blue ) ANO8 . Receptor mediated store depletion was initiated by stimulating the cells with 0 . 5 mM carbachol in Ca2 + - free solution . Cell stimulation was terminated with 10 µM atropine and ER Ca2 + uptake was initiated by perfusing the cells with a solution containing 5 mM Ca2 + . The fluorescence was measured as F / F0 and normalized to the initial fluorescence in the presence of ANO8 . Panel ( C ) shows traces of ER Ca2 + uptake at expanded time scale in the periods marked by rectangular in ( A and B ) and the averaged slopes of ER Ca2 + influx and ( D ) shows the averaged slope of Ca2 + release at the cell periphery ( Peri ) and cell center ( Center ) . In ( C ) the traces were aligned along the Y axis to better show the difference in uptake rate . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B): ER Ca2+ content was measured with ER-GECO1 in the periphery (A) and the center (B) or cells transfected with M3 receptors and with (red, green) or without (black, blue) ANO8. Receptor mediated store depletion was initiated by stimulating the cells with 0.5 mM carbachol in Ca2+-free solution. Cell stimulation was terminated with 10 µM atropine and ER Ca2+ uptake was initiated by perfusing the cells with a solution containing 5 mM Ca2+. The fluorescence was measured as F/F0 and normalized to the initial fluorescence in the presence of ANO8. Panel (C) shows traces of ER Ca2+ uptake at expanded time scale in the periods marked by rectangular in (A and B) and the averaged slopes of ER Ca2+ influx and (D) shows the averaged slope of Ca2+ release at the cell periphery (Peri) and cell center (Center). In (C) the traces were aligned along the Y axis to better show the difference in uptake rate. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( E ) Enhancement by ANO8 of the Co - IP of expressed STIM1 with PMCA4a and SERCA2 and of Orai1 with SERCA2 in resting and store depleted cells . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test .

O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Enhancement by ANO8 of the Co-IP of expressed STIM1 with PMCA4a and SERCA2 and of Orai1 with SERCA2 in resting and store depleted cells. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( F ) Effect of ANO8 expression on the Co - IP of the native SERCA2 and STIM1 in resting and store depleted cells . The columns are the mean ± s . e . m of 3 experiments . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test .

O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Effect of ANO8 expression on the Co-IP of the native SERCA2 and STIM1 in resting and store depleted cells. The columns are the mean±s.e.m of 3 experiments. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( G ) FRET ratio between STIM1 and SERCA2 in response to store depletion and its enhancement by ANO8 . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) FRET ratio between STIM1 and SERCA2 in response to store depletion and its enhancement by ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( H , I ) Simultaneous measurements of ER Ca2 + with ER - GECO1 and PI ( 4 , 5 ) P2 hydrolysis with PHPLCδ - GFP in response to cell stimulation of M3 receptors . Traces are shown in ( h ) and the average slopes are in ( i ) . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H, I) Simultaneous measurements of ER Ca2+ with ER-GECO1 and PI(4,5)P2 hydrolysis with PHPLCδ-GFP in response to cell stimulation of M3 receptors. Traces are shown in (h) and the average slopes are in (i). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( J ) Effect of ANO8 on the Co - IP of the native STIM1 and IP3Rs . The columns are the mean ± s . e . m of 3 experiments . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test .

O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Effect of ANO8 on the Co-IP of the native STIM1 and IP3Rs. The columns are the mean±s.e.m of 3 experiments. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( K ) FRET ratio between expressed STIM1 and IP3R3 in response to store depletion and its enhancement by ANO8 . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) FRET ratio between expressed STIM1 and IP3R3 in response to store depletion and its enhancement by ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( L ) FRET ratio between expressed STIM1 and PMCA4 in response to store depletion and its enhancement by ANO8 . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test .

(L) FRET ratio between expressed STIM1 and PMCA4 in response to store depletion and its enhancement by ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( A - D ) HeLa cells transfected with GFP ( black ) or ANO8 ( red ) were stimulated with 0 . 5 and then 1 µM ATP to activate the native P2Y2 receptors . The oscillations were analyzed in terms of the % of responding cells ( B ) , oscillation frequency ( C ) , and the amplitude of the Ca2 + signal ( D ) . Data information : All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-D) HeLa cells transfected with GFP (black) or ANO8 (red) were stimulated with 0.5 and then 1 µM ATP to activate the native P2Y2 receptors. The oscillations were analyzed in terms of the % of responding cells (B), oscillation frequency (C), and the amplitude of the Ca2+ signal (D). Data information: All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( E - H ) : HeLa cells were treated with scrambled siRNA ( black ) or siANO8 ( red ) and were stimulated with 1 and then 5 µM ATP . The oscillations were analyzed in terms of the % of responding cells ( F ) , oscillations frequency ( G ) , and the amplitude of the Ca2 + signal ( H ) . Data information : All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-H): HeLa cells were treated with scrambled siRNA (black) or siANO8 (red) and were stimulated with 1 and then 5 µM ATP. The oscillations were analyzed in terms of the % of responding cells (F), oscillations frequency (G), and the amplitude of the Ca2+ signal (H). Data information: All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( A ) Total lysates from the indicated cell lines cultured in regular conditions at a steady state were subjected to western blotting . s . e . , short exposure ; l . e . , long exposure . The major Merlin band is indicated by an asterisk . The ratio of mono - ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot . The same lysates were also used in Figure EV1A . Asterisks indicate native Merlin , arrows show ubiquitinated Merlin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O

(A) Total lysates from the indicated cell lines cultured in regular conditions at a steady state were subjected to western blotting. s.e., short exposure; l.e., long exposure. The major Merlin band is indicated by an asterisk. The ratio of mono-ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot. The same lysates were also used in Figure EV1A. Asterisks indicate native Merlin, arrows show ubiquitinated Merlin.


( B ) Total lysates from LN229 cells in which Merlin was or was not knocked out ( KO ) were subjected to western blotting .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

(B) Total lysates from LN229 cells in which Merlin was or was not knocked out (KO) were subjected to western blotting.


LN229 ( C ) cells were detached ( denoted by sus . , referring to suspension ) by trypsinization ( see Methods ) and reseeded ( attach ) for two ( LN229 ) hours . Total lysates from these cells were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . Ubiquitinated Merlin is marked with arrows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O

LN229 (C) cells were detached (denoted by sus., referring to suspension) by trypsinization (see Methods) and reseeded (attach) for two (LN229) hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with arrows.


Met5 - A ( D ) cells were detached ( denoted by sus . , referring to suspension ) by trypsinization ( see Methods ) and reseeded ( attach ) for four ( Met5 - A ) hours . Total lysates from these cells were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . Ubiquitinated Merlin is marked with arrows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O

Met5-A (D) cells were detached (denoted by sus., referring to suspension) by trypsinization (see Methods) and reseeded (attach) for four (Met5-A) hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with arrows.


( E ) LN229 cells transduced with a Merlin or scrambled shRNA were treated with DMSO , ionomycin ( Iono ) or thapsigargin ( TG ) . Total lysates of these cells were subjected to western blotting . The major Merlin band is indicated by an asterisk . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . Ubiquitinated Merlin is marked with an arrow .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O

(E) LN229 cells transduced with a Merlin or scrambled shRNA were treated with DMSO, ionomycin (Iono) or thapsigargin (TG). Total lysates of these cells were subjected to western blotting. The major Merlin band is indicated by an asterisk. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with an arrow.


( F ) LN229 cells treated with DMSO or thapsigargin ( TG ) were lysed and subjected to immunoprecipitation with a Merlin antibody or IgG . The immunoprecipitated products were subjected to western blotting . The number of conjugated ubiquitin ( Ub ) moieties was marked . Tetra - ubiquitinated Merlin is indicated by an arrow .

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O

(F) LN229 cells treated with DMSO or thapsigargin (TG) were lysed and subjected to immunoprecipitation with a Merlin antibody or IgG. The immunoprecipitated products were subjected to western blotting. The number of conjugated ubiquitin (Ub) moieties was marked. Tetra-ubiquitinated Merlin is indicated by an arrow.


( G ) LN229 cells stably transduced with 6x - Histidine - tagged ubiquitin ( His - Ubi ) were treated with DMSO or thapsigargin ( TG ) . These cells were lysed and subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . Both arrowhead and arrow point to the mono - ubiquitinated Merlin . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(G) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were treated with DMSO or thapsigargin (TG). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Both arrowhead and arrow point to the mono-ubiquitinated Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( H ) LN229 cells stably transduced with 6x - Histidine - tagged ubiquitin ( His - Ubi ) were detached or reseeded as in ( C ) . These cells were lysed and subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . Arrowheads and arrows point to the mono - or di - ubiquitinated Merlin ( see a longer exposed blotting results in Figure EV1D ) . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(H) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were detached or reseeded as in (C). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Arrowheads and arrows point to the mono- or di-ubiquitinated Merlin (see a longer exposed blotting results in Figure EV1D). The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( A ) LN229 cells were detached ( denoted by sus . , referring to suspension ) by trypsinization and reseeded ( denoted by attach ) for two hours . Total lysates from these cells were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(A) LN229 cells were detached (denoted by sus., referring to suspension) by trypsinization and reseeded (denoted by attach) for two hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( B ) LN229 cells treated with DMSO , ionomycin ( Iono ) or thapsigargin ( TG ) were lysed and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(B) LN229 cells treated with DMSO, ionomycin (Iono) or thapsigargin (TG) were lysed and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( C ) LN229 cells treated with DMSO or thapsigargin ( TG ) were subjected to active RAC1 pulldown assay followed by western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(C) LN229 cells treated with DMSO or thapsigargin (TG) were subjected to active RAC1 pulldown assay followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


LN229 cells transduced with vector or EGFP - RAC1 ( Q61L ) ( D ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

LN229 cells transduced with vector or EGFP-RAC1(Q61L) (D) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( E ) LN229 cells transduced with vector or Myc - PAK1 ( T423E ) ( E ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(E) LN229 cells transduced with vector or Myc-PAK1(T423E) (E) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( F ) LN229 cells stably transduced with wild - type Merlin or the indicated mutants were treated with DMSO or thapsigargin ( TG ) and analyzed by western blot . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . Native Merlin ( asterisk ) ; ubiquitinated Merlin ( arrow ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O

(F) LN229 cells stably transduced with wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG) and analyzed by western blot. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Native Merlin (asterisk); ubiquitinated Merlin (arrow).


( G ) Merlin - depleted LN229 cells stably transduced with Flag - tagged wild - type Merlin or the indicated mutants were treated with DMSO or thapsigargin ( TG ) . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The lysate and immunoprecipitated products were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(G) Merlin-depleted LN229 cells stably transduced with Flag-tagged wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG). The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( B ) LN229 cells transduced with a pool of four siRNAs against NEDD4L ( + ) or a scrambled siRNA ( - ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(B) LN229 cells transduced with a pool of four siRNAs against NEDD4L (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot.


( C ) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(C) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( D ) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were detached by trypsinization ( see Methods ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(D) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were detached by trypsinization (see Methods) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( E ) LN229 cells stably transduced with 6x - Histidine - tagged Ubiquitin ( His - Ubi ) were then stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA . These cells were detached as in ( D ) , lysed and subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(E) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA. These cells were detached as in (D), lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( F ) HEK293T cells were transfected with indicated genes and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(F) HEK293T cells were transfected with indicated genes and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( G ) HEK293T cells were transfected with indicated genes . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The immunoprecipitated products were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(G) HEK293T cells were transfected with indicated genes. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( H ) LN229 cells were lysed and subjected to immunoprecipitation with a Merlin antibody , a NEDD4L antibody , or IgG . The immunoprecipitated products were subjected to western blotting .

O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(H) LN229 cells were lysed and subjected to immunoprecipitation with a Merlin antibody, a NEDD4L antibody, or IgG. The immunoprecipitated products were subjected to western blotting.


( A ) LN229 cells transduced with a pool of four siRNAs against AMOTL1 ( + ) or a scrambled siRNA ( - ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(A) LN229 cells transduced with a pool of four siRNAs against AMOTL1 (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( B ) LN229 cells stably transduced with 6x - Histidine - tagged Ubiquitin ( His - Ubi ) were then stably transduced with indicated shRNAs against AMOTL1 or a scrambled shRNA control ( sh - Co . ) . These cells were detached , lysed and subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(B) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs against AMOTL1 or a scrambled shRNA control (sh-Co.). These cells were detached, lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( D ) Merlin - depleted LN229 cells stably expressing Flag - HA - tagged Merlin were transduced with scrambled shRNA ( sh - Co . ) or two distinct shRNAs targeting AMOTL1 , treated with DMSO or thapsigargin ( TG ) and subjected to Flag immunoprecipitation followed by western blotting .

O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O

(D) Merlin-depleted LN229 cells stably expressing Flag-HA-tagged Merlin were transduced with scrambled shRNA (sh-Co.) or two distinct shRNAs targeting AMOTL1, treated with DMSO or thapsigargin (TG) and subjected to Flag immunoprecipitation followed by western blotting.


( E ) HEK293T cells were transfected with the indicated constructs . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The lysate and immunoprecipitated products were subjected to western blotting . PY : PPEY motif ; LY : LPTY motif . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(E) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. PY: PPEY motif; LY: LPTY motif. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( F ) Merlin - depleted LN229 cells stably transduced with Flag - tagged wild - type Merlin or the indicated mutants were treated with DMSO or thapsigargin ( TG ) . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The lysate and immunoprecipitated products were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(F) Merlin-depleted LN229 cells stably transduced with Flag-tagged wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG). The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( G ) HEK293T cells were transfected with the indicated constructs . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The lysate and immunoprecipitated products were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(G) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( A ) LN229 cells stably transduced with Flag - tagged wild - type Merlin or the S518D mutant were treated with thapsigargin and subjected to immunoprecipitation with a Flag antibody and followed by SDS - PAGE . Outlined gel pieces were analyzed by mass spectrometry . ( B ) Numbers of unique peptides of Merlin or Ubiquitin from each outlined gel pieces in ( A ) are shown .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O

(A) LN229 cells stably transduced with Flag-tagged wild-type Merlin or the S518D mutant were treated with thapsigargin and subjected to immunoprecipitation with a Flag antibody and followed by SDS-PAGE. Outlined gel pieces were analyzed by mass spectrometry. (B) Numbers of unique peptides of Merlin or Ubiquitin from each outlined gel pieces in (A) are shown.


( D ) and ( E ) Mass spectrometry spectrums of the indicated peptides from each immunoprecipitated product are shown .

O O O O O O O O O O O O O O O O O O O O O

(D) and (E) Mass spectrometry spectrums of the indicated peptides from each immunoprecipitated product are shown.


( F ) Merlin - depleted LN229 cells expressing His - Ubiquitin were stably transduced with the indicated HA - tagged Merlin forms or an empty vector . These cells treated with DMSO or thapsigargin were subjected to nickel affinity purification in a denaturing step followed by western blotting for HA . Arrows indicate mono - ubiquitinated Merlin . The red asterisk indicates native Merlin and black asterisks indicate non - specific signals . The ratio of mono - ubiquitinated to native Merlin in each lane of the input blot was quantified by Image J and is shown under the blot .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(F) Merlin-depleted LN229 cells expressing His-Ubiquitin were stably transduced with the indicated HA-tagged Merlin forms or an empty vector. These cells treated with DMSO or thapsigargin were subjected to nickel affinity purification in a denaturing step followed by western blotting for HA. Arrows indicate mono-ubiquitinated Merlin. The red asterisk indicates native Merlin and black asterisks indicate non-specific signals. The ratio of mono-ubiquitinated to native Merlin in each lane of the input blot was quantified by Image J and is shown under the blot.


( G ) Merlin - depleted LN229 cells were stably transduced with an empty vector , Flag - tagged wild - type ( WT ) Merlin or its K396R mutant . These cells were detached ( S , suspension ) by trypsinization using the procedure described in the Methods section and reseeded ( A , attached ) for two hours . Cell lysates were subjected to immunoprecipitation with a Flag antibody followed by western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(G) Merlin-depleted LN229 cells were stably transduced with an empty vector, Flag-tagged wild-type (WT) Merlin or its K396R mutant. These cells were detached (S, suspension) by trypsinization using the procedure described in the Methods section and reseeded (A, attached) for two hours. Cell lysates were subjected to immunoprecipitation with a Flag antibody followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( H ) Merlin - depleted LN229 cells stably transduced with wild - type ( WT ) Merlin or its K396R mutant were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(H) Merlin-depleted LN229 cells stably transduced with wild-type (WT) Merlin or its K396R mutant were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( I ) Merlin - depleted LN229 cells stably transduced with HA - tagged wild - type ( WT ) Merlin or its K396R mutant were treated as described in ( G ) and subjected to western blotting . The ratio of mono - ubiquitinated to native null in each lane was quantified by Image J and is shown under the blot .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Merlin-depleted LN229 cells stably transduced with HA-tagged wild-type (WT) Merlin or its K396R mutant were treated as described in (G) and subjected to western blotting. The ratio of mono-ubiquitinated to native null in each lane was quantified by Image J and is shown under the blot.


( A ) LN229 cells were treated with DMSO or thapsigargin ( TG ) for the indicated time and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(A) LN229 cells were treated with DMSO or thapsigargin (TG) for the indicated time and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( B ) Merlin - depleted LN229 cells were reconstituted with wild type ( WT ) Merlin or its mutants , treated with DMSO or thapsigargin ( TG ) , and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(B) Merlin-depleted LN229 cells were reconstituted with wild type (WT) Merlin or its mutants, treated with DMSO or thapsigargin (TG), and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( C ) Merlin - depleted LN229 cells were reconstituted with wild type ( WT ) Merlin or its mutants , treated with DMSO or thapsigargin ( TG ) , and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

(C) Merlin-depleted LN229 cells were reconstituted with wild type (WT) Merlin or its mutants, treated with DMSO or thapsigargin (TG), and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( D ) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L ( + ) or a scrambled siRNA ( - ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O

(D) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting.


( E ) Merlin - depleted LN229 cells were stably transduced with empty vector , Flag - tagged wild - type ( WT ) Merlin , or its K396R mutant . These cells were detached ( S , suspension ) by trypsinization using the procedure described in Methods and reseeded ( A , attached ) for two hours . Cell lysates were subjected to western blotting .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Merlin-depleted LN229 cells were stably transduced with empty vector, Flag-tagged wild-type (WT) Merlin, or its K396R mutant. These cells were detached (S, suspension) by trypsinization using the procedure described in Methods and reseeded (A, attached) for two hours. Cell lysates were subjected to western blotting.


( F ) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L ( + ) or a scrambled siRNA ( - ) were detached ( Susp . , suspension ) by trypsinization using the procedure described in Methods and reseeded ( Attach , attached ) for two hours . Total lysates from these cells were subjected to western blotting .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L (+) or a scrambled siRNA (-) were detached (Susp., suspension) by trypsinization using the procedure described in Methods and reseeded (Attach, attached) for two hours. Total lysates from these cells were subjected to western blotting.


( G ) LN229 cells transfected with indicated siRNAs were seeded for 3 hours . CTGF or CYR61 mRNA in these cells was quantified by q - RT - PCR . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 . Mean ± s . e . m , N = 4 biological repeats , unpaired t - test .

O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) LN229 cells transfected with indicated siRNAs were seeded for 3 hours. CTGF or CYR61 mRNA in these cells was quantified by q-RT-PCR. *P<0.05, **P<0.01, ****P<0.0001. Mean±s.e.m, N=4 biological repeats, unpaired t-test.


( A ) LN229 cells stably transduced withHA - Lats1 ( two leftmost panels ) or vector only ( right panel ) were treated with DMSO or thapsigargin ( TG ) and subjected to PLA using HA and Merlin antibodies . Scale bar = 10 μm . ( B ) PLA signals ( dots ) in each cell from the results in ( A ) were quantified . Mean ± s . e . m , two - way ANOVA . * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each data point represents an image field containing an average of 10 cells . N = 4 - 9 images for each condition as indicated . All images were collected from one experiment . Two independent experiments were performed and gave similar results .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) LN229 cells stably transduced withHA-Lats1 (two leftmost panels) or vector only (right panel) were treated with DMSO or thapsigargin (TG) and subjected to PLA using HA and Merlin antibodies. Scale bar = 10 μm. (B) PLA signals (dots) in each cell from the results in (A) were quantified. Mean±s.e.m, two-way ANOVA. *P<0.05, ***P<0.001, ****P<0.0001. Each data point represents an image field containing an average of 10 cells. N=4-9 images for each condition as indicated. All images were collected from one experiment. Two independent experiments were performed and gave similar results.


( C ) Merlin - depleted LN229 cells were stably transduced with HA - tagged wild - type ( WT ) Merlin or its mutants . These cells were treated with thapsigargin ( TG ) for 15 minutes and subjected to PLA using HA and Lats1 antibodies . Scale bar = 20 μm . ( D ) PLA signals ( dots ) in each cell from the results in ( C ) were quantified . Mean ± s . e . m , Ordinary one - way ANOVA . * * * * P < 0 . 0001 . Each data point represents an image field containing an average of 10 cells . NVector = 8 , NWT = 16 , NK396R = 18 , NK159R = 17 images . All images were collected from one experiment . Two independent experiments were performed and gave similar results .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Merlin-depleted LN229 cells were stably transduced with HA-tagged wild-type (WT) Merlin or its mutants. These cells were treated with thapsigargin (TG) for 15 minutes and subjected to PLA using HA and Lats1 antibodies. Scale bar = 20 μm. (D) PLA signals (dots) in each cell from the results in (C) were quantified. Mean±s.e.m, Ordinary one-way ANOVA. ****P<0.0001. Each data point represents an image field containing an average of 10 cells. NVector=8, NWT=16, NK396R=18, NK159R=17 images. All images were collected from one experiment. Two independent experiments were performed and gave similar results.


( E ) Merlin - depleted LN229 cells stably transduced with empty vector , Flag - tagged wild - type ( WT ) Merlin , or its K396R mutant were subjected to immunoprecipitation with a Flag antibody followed by western blotting .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

(E) Merlin-depleted LN229 cells stably transduced with empty vector, Flag-tagged wild-type (WT) Merlin, or its K396R mutant were subjected to immunoprecipitation with a Flag antibody followed by western blotting.


( F ) HEK293T cells were transfected with indicated proteins and subjected to immunoprecipitation with a Flag antibody . The lysates and immunoprecipitated products were subjected to western blotting .

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(F) HEK293T cells were transfected with indicated proteins and subjected to immunoprecipitation with a Flag antibody. The lysates and immunoprecipitated products were subjected to western blotting.


( G ) Merlin - depleted LN229 cells were stably transduced by HA - tagged Merlin . These cells were then transduced with a pool of four siRNAs against NEDD4L or a scrambled control siRNA and treated with DMSO or thapsigargin ( TG ) . PLA was performed in these cells using HA and Lats1 antibodies , and the PLA signals ( dots , showed in Figure EV5E ) in each cell were quantified . Mean ± s . e . m , two - way ANOVA . * * * * P < 0 . 0001 . Each data point represents an image field containing averagely 10 cells . N = 12 images in all conditions , except for Nsi - Control : TG = 13 images . All images were collected from one experiment . Two independent experiments were performed and showed similar results .

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(G) Merlin-depleted LN229 cells were stably transduced by HA-tagged Merlin. These cells were then transduced with a pool of four siRNAs against NEDD4L or a scrambled control siRNA and treated with DMSO or thapsigargin (TG). PLA was performed in these cells using HA and Lats1 antibodies, and the PLA signals (dots, showed in Figure EV5E) in each cell were quantified. Mean±s.e.m, two-way ANOVA. ****P<0.0001. Each data point represents an image field containing averagely 10 cells. N=12 images in all conditions, except for Nsi-Control:TG=13 images. All images were collected from one experiment. Two independent experiments were performed and showed similar results.


( H ) LN229 cells treated with DMSO or thapsigargin ( TG ) were subjected to cytosolic ( C ) / membrane ( M ) fractionation followed by western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

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(H) LN229 cells treated with DMSO or thapsigargin (TG) were subjected to cytosolic (C)/membrane (M) fractionation followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( A ) Met5 - A cells stably transduced with the indicated shRNAs targeting Merlin or a scrambled shRNA were subjected to the BrdU incorporation assay . Scale bar = 40 μm . ( B ) The percentage of BrdU positive cells from ( A ) was quantified . Mean ± s . e . m , N = 3 biological repeats , paired t - test . * * P < 0 . 01 , * * * P < 0 . 001 .

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(A) Met5-A cells stably transduced with the indicated shRNAs targeting Merlin or a scrambled shRNA were subjected to the BrdU incorporation assay. Scale bar = 40 μm. (B) The percentage of BrdU positive cells from (A) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.01, ***P<0.001.


( C ) Met5 - A cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were subjected to the BrdU incorporation assay . Scale bar = 40 μm . ( D ) The percentage of BrdU positive cells from ( C ) was quantified . Mean ± s . e . m , N = 3 biological repeats , paired t - test . * * P < 0 . 01 , * * * P < 0 . 001 .

(C) Met5-A cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were subjected to the BrdU incorporation assay. Scale bar = 40 μm. (D) The percentage of BrdU positive cells from (C) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.01, ***P<0.001.


( E ) Meso - 33 cells transduced with vector or the indicated Merlin forms were subjected to the BrdU incorporation assay . Scale bar = 40 μm . ( F ) The percentage of BrdU positive cells from ( E ) was quantified . Mean ± s . e . m , N = 3 biological repeats , ordinary one - way ANOVA . * P < 0 . 05 , * * P < 0 . 01 .

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(E) Meso-33 cells transduced with vector or the indicated Merlin forms were subjected to the BrdU incorporation assay. Scale bar = 40 μm. (F) The percentage of BrdU positive cells from (E) was quantified. Mean±s.e.m, N=3 biological repeats, ordinary one-way ANOVA. *P<0.05, **P<0.01.


( G ) Meso - 33 cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were then transduced with vector or Merlin prior to the BrdU incorporation assay . The percentage of BrdU positive cells was quantified . Mean ± s . e . m , N = 3 biological repeats , two - way ANOVA .

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(G) Meso-33 cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were then transduced with vector or Merlin prior to the BrdU incorporation assay. The percentage of BrdU positive cells was quantified. Mean±s.e.m, N=3 biological repeats, two-way ANOVA.


( H ) FC - 1801 cells stably transduced with vector or the indicated Merlin forms were subjected to the sphere formation assay . Scale bar = 500 μm . ( I ) The number of spheres in each well from ( H ) was quantified . Mean ± s . e . m , N = 3 biological repeats , paired t - test . * * P < 0 . 05 .

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(H) FC-1801 cells stably transduced with vector or the indicated Merlin forms were subjected to the sphere formation assay. Scale bar = 500 μm. (I) The number of spheres in each well from (H) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.05.


( J ) FC - 1801 cells stably transduced with vector or the indicated Merlin forms were subcutaneously injected into nude mice . Tumor sizes in each group were quantified and plotted . Mean ± s . e . m , NVector = 8 mice , NWT = 10 mice , NK396R = 10 mice , NS518D = 8 mice , two - way ANOVA . P values of the last measurements ( day 37 after injection ) are shown . * P < 0 . 05 , * * P < 0 . 01 . ( K ) Tumors were collected when the vector group in ( J ) reached the end point ( day 37 after injection ) .

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(J) FC-1801 cells stably transduced with vector or the indicated Merlin forms were subcutaneously injected into nude mice. Tumor sizes in each group were quantified and plotted. Mean±s.e.m, NVector=8 mice, NWT=10 mice, NK396R=10 mice, NS518D=8 mice, two-way ANOVA. P values of the last measurements (day 37 after injection) are shown. *P<0.05, **P<0.01. (K) Tumors were collected when the vector group in (J) reached the end point (day 37 after injection).


Confocal microscopy of IBA - 1 ( green staining ) immunohistochemistry of RPE flatmounts ( RPE autofluorescence visible as orange due to its autofluorescence in the red and green channel ) from a healthy donor ( A ) , a geographic atrophy lesion ( B ) , and large drusen ( C and D ) . ( A , B , D ) : orthogonal Z - stack projection ; ( C ) : oblique Z - stack projection and dissecting microscope appearance of postmortem large drusen after removal of the overlaying retina ( inset ) .

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Confocal microscopy of IBA-1 (green staining) immunohistochemistry of RPE flatmounts (RPE autofluorescence visible as orange due to its autofluorescence in the red and green channel) from a healthy donor (A), a geographic atrophy lesion (B), and large drusen (C and D). (A, B, D): orthogonal Z-stack projection; (C): oblique Z-stack projection and dissecting microscope appearance of postmortem large drusen after removal of the overlaying retina (inset).


Double - labeling on the subretinal side of the retina ( to avoid masking by RPE autofluorescence ) of IBA - 1 + ( E , green fluorescence ) and CD18 ( F , red fluorescence ; G , merge ) .

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Double-labeling on the subretinal side of the retina (to avoid masking by RPE autofluorescence) of IBA-1+ (E, green fluorescence) and CD18 (F, red fluorescence; G, merge).


Orthogonal and lateral Z - stack of a subretinal IBA - 1 + ( green fluorescence ) MPs adjacent to the RPE ( orange autofluorescence ) in the vicinity of a large drusen .

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Orthogonal and lateral Z-stack of a subretinal IBA-1+ (green fluorescence) MPs adjacent to the RPE (orange autofluorescence) in the vicinity of a large drusen.


APOE ( A ) and IBA - 1 ( B , merge in C ) double - labeling on paraffin sections of surgical specimens of human tonsils .

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APOE (A) and IBA-1 (B, merge in C) double-labeling on paraffin sections of surgical specimens of human tonsils.


APOE ( D ) and IBA - 1 ( E , merge in F ) double - labeling on the subretinal aspect of a large drusen overlaying retina ( to avoid specific signal masking by the RPE orange autofluorescence ) .

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APOE (D) and IBA-1 (E, merge in F) double-labeling on the subretinal aspect of a large drusen overlaying retina (to avoid specific signal masking by the RPE orange autofluorescence).


APOE ( red staining ) immunohistochemistry of control donor tissue ( G ) and adjacent to GA lesion ( H ) .

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APOE (red staining) immunohistochemistry of control donor tissue (G) and adjacent to GA lesion (H).


IBA - 1 ( green staining , RPE far - red autofluorescence ) double - labeling of the GA lesion shown in ( H ) . Representative images from five donor eyes with large drusen , five donor eyes with GA , and three healthy donors between 70 and 89 years old .

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IBA-1 (green staining, RPE far-red autofluorescence) double-labeling of the GA lesion shown in (H). Representative images from five donor eyes with large drusen, five donor eyes with GA, and three healthy donors between 70 and 89 years old.


Immunohistochemistry of APOE ( red ) and IBA - 1 ( green ) on a section ( A , blue Hoechst ) and the subretinal side of a retinal flatmount ( B and C ) from a 12 - month - old Cx3cr1GFP / GFP mouse ( representative of 3 independent experiments , experiments omitting the primary antibody immunostaining served as negative controls ) . ONL : outer nuclear layer ; RPE : retinal pigment epithelium .

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Immunohistochemistry of APOE (red) and IBA-1 (green) on a section (A, blue Hoechst) and the subretinal side of a retinal flatmount (B and C) from a 12-month-old Cx3cr1GFP/GFP mouse (representative of 3 independent experiments, experiments omitting the primary antibody immunostaining served as negative controls). ONL: outer nuclear layer; RPE: retinal pigment epithelium.


Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP monocytes cultured for 24 h in contact with POS of an overlaying retinal explant ( n = 4 / group per experiment ; Mann - Whitney U - test , * P = 0 . 0286 , the experiment was repeated twice with similar results ) . Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP FACS - sorted microglial cells , freshly extracted from adult brain ( n = 4 - 5 / group ; Mann - Whitney U - test , C57BL / 6J versus Cx3cr1GFP / GFP * P = 0 . 0159 ) . Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP peritoneal macrophages cultured for 24 h with CX3CL1 . ( n = 4 / group per experiment ; Mann - Whitney U - test , * P = 0 . 0286 . The experiment was repeated twice with similar results ) .

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Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP monocytes cultured for 24 h in contact with POS of an overlaying retinal explant (n = 4/group per experiment; Mann-Whitney U-test, *P = 0.0286, the experiment was repeated twice with similar results).Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP FACS-sorted microglial cells, freshly extracted from adult brain (n = 4-5/group; Mann-Whitney U-test, C57BL/6J versus Cx3cr1GFP/GFP *P = 0.0159).Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP peritoneal macrophages cultured for 24 h with CX3CL1. (n = 4/group per experiment; Mann-Whitney U-test, *P = 0.0286. The experiment was repeated twice with similar results).


APOE Western blot analysis of equivalent amounts of supernatant protein from CX3CL1 - exposed C57BL / 6J and Cx3cr1GFP / GFP peritoneal macrophages at 24 h . Soluble Mer receptor tyrosine kinase that is released constitutively from cultured macrophages served as a loading control . The experiment was repeated twice with similar results .

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APOE Western blot analysis of equivalent amounts of supernatant protein from CX3CL1-exposed C57BL/6J and Cx3cr1GFP/GFP peritoneal macrophages at 24 h. Soluble Mer receptor tyrosine kinase that is released constitutively from cultured macrophages served as a loading control. The experiment was repeated twice with similar results.