Figure 9A - B Growth curves of Salmonella glnA : : glnAWT , glnA : : glnAY398S , glnA : : glnAY398F , glnA : : glnA2KQY398F , glnA : : glnA2KQY398S , glnA : : glnA2KQY398S - gdhA : : gdhAK128Q , and glnA : : glnA2KQY398S - gdhA : : gdhAK128R strains were measured in minimal medium with 100 mM ammonium and either 5 mM ( A ) or 50 mM glucose ( B ) in the media . data are pooled from the three independent experimentsE - I The bacterial loads of the faeces ( E and F ) , caecum ( G ) , spleen ( H ) , and liver ( I ) of C57BL / 6 mouse littermates infected with Salmonella strains glnA : : glnAWT ( n = 4 ) , glnA : : glnAY398S ( n = 4 ) , glnA : : glnA2KQY398S ( n = 4 ) , glnA : : glnA2KQY398SgdhA : : gdhAK128Q ( n = 4 ) , and glnA : : glnA2KQY398S - gdhA : : gdhAK128R ( n = 4 ) were measured by counting colony - forming units ( CFU ) in cultures of serially diluted homogenates of organs on MacConkey agar plates . Each symbol represents an independent biological replicates ( E - I )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9A-B Growth curves of Salmonella glnA::glnAWT, glnA::glnAY398S, glnA::glnAY398F, glnA::glnA2KQY398F, glnA::glnA2KQY398S, glnA::glnA2KQY398S-gdhA::gdhAK128Q, and glnA::glnA2KQY398S-gdhA::gdhAK128R strains were measured in minimal medium with 100 mM ammonium and either 5 mM (A) or 50 mM glucose (B) in the media. data are pooled from the three independent experimentsE-I The bacterial loads of the faeces (E and F), caecum (G), spleen (H), and liver (I) of C57BL/6 mouse littermates infected with Salmonella strains glnA::glnAWT (n=4), glnA::glnAY398S (n=4), glnA::glnA2KQY398S (n=4), glnA::glnA2KQY398SgdhA::gdhAK128Q (n=4), and glnA::glnA2KQY398S-gdhA::gdhAK128R (n=4) were measured by counting colony-forming units (CFU) in cultures of serially diluted homogenates of organs on MacConkey agar plates. Each symbol represents an independent biological replicates (E-I)


Figure 1 ( E ) Confirmation of PARylated proteins with an in vivo PARylation assay . FLAG - tagged candidate proteins were expressed in protoplasts , and immunoprecipitated by α - FLAG antibody after feeding protoplasts with 32P - NAD + . The immunoprecipitated proteins were separated in 10 % SDS - PAGE and detected by autoradiography ( top panel ) . The input of FLAG - tagged proteins is shown in an α - FLAGimmunoblot ( middle panel ) , and the protein loading control is shown by Ponceau S staining for RuBisCo ( RBC ) ( bottom panel ) . The experiments were repeated three times with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(E) Confirmation of PARylated proteins with an in vivo PARylation assay. FLAG-tagged candidate proteins were expressed in protoplasts, and immunoprecipitated by α-FLAG antibody after feeding protoplasts with 32P-NAD+. The immunoprecipitated proteins were separated in 10% SDS-PAGE and detected by autoradiography (top panel). The input of FLAG-tagged proteins is shown in an α-FLAGimmunoblot (middle panel), and the protein loading control is shown by Ponceau S staining for RuBisCo (RBC) (bottom panel). The experiments were repeated three times with similar results.


Figure 2 ( B ) The DDL transcripts and growth phenotype of ddl - 6 and ddl - 7 . DDL transcripts were detected in fully expanded leaves of four - week - old plants by qRT - PCR ( left panel ) . The expression of DDL in WT Col - 0 was set as 1 . Plants grown under 12 - hr light / 12 - hr dark condition for 4 weeks are shown ( right panel ) . Bar = 1cm . ( C ) ddl - 6 and ddl - 7 are more susceptible to Pst DC3000 infections . Leaves of four - week - old plants were inoculated with Pst DC3000 at OD600 = 5x10 - 4 . Bacterial numbers were counted at 0 and 3 dpi . Leaf pictures were taken at 3 dpi . ( D ) The ddl - 1 mutant in the Ws background is more susceptible to Pst DC3000 infection . ( E ) ddl - 6 is more susceptible to Psminfection . Leaves of four - week - old plants were inoculated with Psmat OD600 = 5x10 - 4 . ( F ) Increased bacterial growth of Pst DC3000 hrcC in ddl - 6 . Leaves of four - week - old plants were inoculated with Pst DC3000 hrcC at OD600 = 5x10 - 4 . ( G ) Increased bacterial growth of Psp in ddl - 6 . Leaves of four - week - old plants were inoculated with Psp at OD600 = 5x10 - 4 . ( H ) Complementation of DDL in disease resistance . Two independent T3 homozygous lines carrying pDDL : : DDL - FLAG ( W7 and W18 ) in the ddl - 6 background were assayed for the susceptibility to Pst DC3000 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) The DDL transcripts and growth phenotype of ddl-6 and ddl-7. DDL transcripts were detected in fully expanded leaves of four-week-old plants by qRT-PCR (left panel). The expression of DDL in WT Col-0 was set as 1. Plants grown under 12-hr light/12-hr dark condition for 4 weeks are shown (right panel). Bar=1cm.(C) ddl-6 and ddl-7 are more susceptible to Pst DC3000 infections. Leaves of four-week-old plants were inoculated with Pst DC3000 at OD600=5x10-4. Bacterial numbers were counted at 0 and 3 dpi. Leaf pictures were taken at 3 dpi.(D) The ddl-1 mutant in the Ws background is more susceptible to Pst DC3000 infection.(E) ddl-6 is more susceptible to Psminfection. Leaves of four-week-old plants were inoculated with Psmat OD600=5x10-4.(F) Increased bacterial growth of Pst DC3000 hrcC in ddl-6. Leaves of four-week-old plants were inoculated with Pst DC3000 hrcC at OD600=5x10-4.(G) Increased bacterial growth of Psp in ddl-6. Leaves of four-week-old plants were inoculated with Psp at OD600=5x10-4.(H) Complementation of DDL in disease resistance. Two independent T3 homozygous lines carrying pDDL::DDL-FLAG (W7 and W18) in the ddl-6 background were assayed for the susceptibility to Pst DC3000.


Figure 3 ( A ) flg22 - induced callose deposition is reduced in ddl - 6 and parp1 , 2 . Leaves of four - week - old plants were inoculated with 0 . 5 µM flg22 for 20 hr or hrcC at OD600 = 0 . 2 for 24 hr and stained with aniline blue . Callose deposits were visualized under UV light and quantified by Image J . The data are shown as mean ± SE ( n = 12 ) . Bar = 100 µm . ( B ) The hrcC - mediated induction of some late responsive genes is compromised in the ddl - 6 and parp1 , 2 mutants . Fully expanded leaves of four - week - old plants were hand - inoculated with hrcC ( OD600 = 0 . 5 ) , and collected at 0 , 3 and 24 hpi for qRT - PCR . The data are shown as mean ± SE from three independent repeats . ( C ) Pst DC3000 - induced PR1and PR5transcripts are blocked in ddl - 6 and parp1 , 2 . Fully expanded leaves of four - week - old plants were inoculated with Pst DC3000 ( OD600 = 0 . 01 ) , and collected at 0 , 8 and 24 hpi for qRT - PCR . The data are shown as mean ± SE from three independent repeats . ( D ) flg22 - induced MAPK activation in Col - 0 and ddl - 6 . Ten - day - old seedlings were treated with 100 nM flg22 and collected at the indicted time points . MAPK activation was analyzed by an immunoblot ( IB ) with - pERK antibody ( top panel ) , and the protein loading is shown by Ponceau S staining for RuBisCo ( RBC ) ( bottom panel ) . ( E ) flg22 - induced ROS in Col - 0 and ddl - 6 . Leaf discs from four - week - old plants were assayed for ROS production upon 100 nM flg22 treatment over 30 min . The data are show as mean ± SE ( n = 24 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) flg22-induced callose deposition is reduced in ddl-6 and parp1,2. Leaves of four-week-old plants were inoculated with 0.5 µM flg22 for 20 hr or hrcC at OD600=0.2 for 24 hr and stained with aniline blue. Callose deposits were visualized under UV light and quantified by Image J. The data are shown as mean ± SE (n=12). Bar=100 µm.(B) The hrcC-mediated induction of some late responsive genes is compromised in the ddl-6 and parp1,2 mutants. Fully expanded leaves of four-week-old plants were hand-inoculated with hrcC(OD600=0.5), and collected at 0, 3 and 24 hpi for qRT-PCR. The data are shown as mean ± SE from three independent repeats.(C) Pst DC3000-induced PR1and PR5transcripts are blocked in ddl-6 and parp1,2. Fully expanded leaves of four-week-old plants were inoculated with Pst DC3000 (OD600=0.01), and collected at 0, 8 and 24 hpi for qRT-PCR. The data are shown as mean ± SE from three independent repeats.(D) flg22-induced MAPK activation in Col-0 and ddl-6. Ten-day-old seedlings were treated with 100 nM flg22 and collected at the indicted time points. MAPK activation was analyzed by an immunoblot (IB) with -pERK antibody (top panel), and the protein loading is shown by Ponceau S staining for RuBisCo (RBC) (bottom panel).(E) flg22-induced ROS in Col-0 and ddl-6. Leaf discs from four-week-old plants were assayed for ROS production upon 100 nM flg22 treatment over 30 min. The data are show as mean± SE (n=24).


Figure 4 ( A ) PARP2 and DDL association in Arabidopsis protoplasts . PARP2 - HA and DDL - FLAG were transiently expressed in Col - 0protoplasts , immunoprecipitated with α - FLAG antibody ( IP : - FLAG ) , and immunoblotted with α - HA ( IB : α - HA ) or α - FLAG antibody ( IB : - FLAG ) ( top two panels ) . The protein inputs are shown with immunoblotting before immunoprecipitation ( bottom two panels ) . Protoplasts were treated without or with 100 nM flg22 for 30 min . ( B ) PARP2 and DDL association in N . benthamiana . PARP2 - FLAG and DDL - HA were transiently co - expressed in leaves of N . benthamiana . The samples were collected at 2 dpi for Co - IP assay with 100nM flg22 treatment for 1 hr . ( C ) PARP2 and DDL association with BiFC assay . Different combinations of constructs were transiently expressed in Arabidopsis Col - 0 protoplasts and the fluorescence signals were observed with a confocal microscope . An NLS - RFP construct was co - transfected to show the nuclear signals . Bar = 10 µm . ( D ) PARP2 and DDL interaction in vitro . GST - PARP2 protein immobilized on glutathione beads was used to pull - downHIS - SUMO - DDL - HA proteins ( left panels ) . A PARylation reaction was performed prior to the pull - down assay ( the third lane of left panels ) . The PARylated PARP2 and DDL were detected with α - PAR antibody ( right panels ) . ( E ) In vitro PARylation of DDL by PARP2 . Immunoprecipitated DDL - FLAG proteins from Arabidopsis protoplasts were incubated with GST - PARP2 in a PARylation reaction containing Biotin - NAD + . The reactions in lane 4 and 5 contained 1 mM 3 - AB or GST - PARG1 respectively . PARylated proteins were detected by streptavidin - HRP antibody ( Strep ) , which recognizes Biotin - labelled PAR .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) PARP2 and DDL association in Arabidopsis protoplasts. PARP2-HA and DDL-FLAG were transiently expressed in Col-0protoplasts, immunoprecipitated with α-FLAG antibody (IP: -FLAG), and immunoblotted with α-HA (IB: α-HA) or α-FLAG antibody (IB: -FLAG) (top two panels). The protein inputs are shown with immunoblotting before immunoprecipitation (bottom two panels). Protoplasts were treated without or with 100 nM flg22 for 30 min.(B) PARP2 and DDL association in N. benthamiana. PARP2-FLAG and DDL-HA were transiently co-expressed in leaves of N. benthamiana. The samples were collected at 2 dpi for Co-IP assay with 100nM flg22 treatment for 1 hr.(C) PARP2 and DDL association with BiFC assay. Different combinations of constructs were transiently expressed in Arabidopsis Col-0 protoplasts and the fluorescence signals were observed with a confocal microscope. An NLS-RFP construct was co-transfected to show the nuclear signals. Bar=10 µm.(D) PARP2 and DDL interaction in vitro. GST-PARP2 protein immobilized on glutathione beads was used to pull-downHIS-SUMO-DDL-HA proteins (left panels). A PARylation reaction was performed prior to the pull-down assay (the third lane of left panels). The PARylated PARP2 and DDL were detected with α-PAR antibody (right panels).(E) In vitro PARylation of DDL by PARP2. Immunoprecipitated DDL-FLAG proteins from Arabidopsis protoplasts were incubated with GST-PARP2 in a PARylation reaction containing Biotin-NAD+. The reactions in lane 4 and 5 contained 1 mM 3-AB or GST-PARG1 respectively. PARylated proteins were detected by streptavidin-HRP antibody (Strep), which recognizes Biotin-labelled PAR.


Figure 5 ( A ) flg22 stimulates DDL PARylation . DDL - FLAG was transiently expressed in Arabidopsis protoplasts fed with 32P - NAD + . Protoplasts were treated without or with 100 nM flg22 for 30 min . PARylated DDL was immunoprecipitated with α - FLAG affinity beads , separated on 10 % SDS - PAGE and visualized by autoradiography . PAR ( DDL ) indicates PARylated DDL proteins . A representative result from three repeats is shown . ( B - C ) DDL PARylation is reduced in parp1 , parp2 and parp1 , 2 mutants . Protoplasts isolated from parp1or parp2 single mutants ( B ) , or parp1 , 2 double mutants ( C ) were used for in vivo PARylation assay with 32P - NAD + . Protoplasts were treated with 100 nM flg22 for 30 min . The experiments were repeated twice with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) flg22 stimulates DDL PARylation. DDL-FLAG was transiently expressed in Arabidopsis protoplasts fed with 32P-NAD+. Protoplasts were treated without or with 100 nM flg22 for 30 min. PARylated DDL was immunoprecipitated with α-FLAG affinity beads, separated on 10% SDS-PAGE and visualized by autoradiography. PAR(DDL) indicates PARylated DDL proteins. A representative result from three repeats is shown.(B-C) DDL PARylation is reduced in parp1, parp2 and parp1,2 mutants. Protoplasts isolated from parp1or parp2 single mutants (B), or parp1,2 double mutants (C) were used for in vivo PARylation assay with 32P-NAD+. Protoplasts were treated with 100 nM flg22 for 30 min. The experiments were repeated twice with similar results.


Figure 6 ( A ) DDL - N but not DDL - C associates with PARP2 . FLAG - tagged DDL - N or DDL - C was co - expressed with PARP2 - HA in protoplasts for Co - IP assay . Protoplasts were treated without or with 100 nM flg22 for 30 min . ( B ) in vivo PARylation of DDL - N or DDL - C in protoplasts fed with 32P - NAD + . PARylation was visualized with autoradiography . ( C ) In vitro PARylation of DDL - N or DDL - C . FLAG - tagged DDL - N or DDL - C was expressed and immunoprecipitated from Arabidopsis protoplasts and subjected to in vitro PARylation by GST - PARP2 with Biotin - NAD + . PARylated proteins were detected by streptavidin - HRP antibody . ( F ) DDL12E mutants substantially diminish its PARylation in vivo . In vivo PARylation of DDL12E1 or DDL12E2 was performed using Arabidopsis protoplasts fed with 32P - NAD + . ( G ) Reduced association of DDL12E1 with PARP2 in protoplasts . Protoplasts were treated with 100 nm flg22 for 30 min before Co - IP assay .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) DDL-N but not DDL-C associates with PARP2. FLAG-tagged DDL-N or DDL-C was co-expressed with PARP2-HA in protoplasts for Co-IP assay. Protoplasts were treated without or with 100 nM flg22 for 30 min.(B) in vivo PARylation of DDL-N or DDL-C in protoplasts fed with 32P-NAD+. PARylation was visualized with autoradiography.(C) In vitro PARylation of DDL-N or DDL-C. FLAG-tagged DDL-N or DDL-C was expressed and immunoprecipitated from Arabidopsis protoplasts and subjected to in vitro PARylation by GST-PARP2 with Biotin-NAD+. PARylated proteins were detected by streptavidin-HRP antibody.(F) DDL12E mutants substantially diminish its PARylation in vivo. In vivo PARylation of DDL12E1 or DDL12E2 was performed using Arabidopsis protoplasts fed with 32P-NAD+.(G) Reduced association of DDL12E1 with PARP2 in protoplasts. Protoplasts were treated with 100 nm flg22 for 30 min before Co-IP assay.


Figure 7 ( A ) Phenotype of plants complemented with WT DDL or DDL12E1 . Four - week - old plants of two independent transgenic lines of pDDL : : DDL - FLAG ( W7 and W18 ) or pDDL : : DDL12E1 - FLAG ( E11 and E15 ) were photographed . Bar = 1cm . ( A ) Phenotype of plants complemented with WT DDL or DDL12E1 . Four - week - old plants of two independent transgenic lines of pDDL : : DDL - FLAG ( W7 and W18 ) or pDDL : : DDL12E1 - FLAG ( E11 and E15 ) . The expression of transgene was determined by an α - FLAGimmunoblot ( bottom ) . ( B ) Susceptibility of complementation lines to Pst DC3000 infections . Four - week - old transgenic plants were inoculated with Pst DC3000 ( OD600 = 5x10 - 4 ) and the bacterial number was determined at 0 and 3 dpi . ( C ) PR1 and PR5 induction in transgenic lines . Leaves of four - week - old plants were inoculated with Pst DC3000 ( OD600 = 0 . 01 ) , and collected at 0 , and 24 hpi for qRT - PCR analysis . The data in B and C are shown as mean ± SD ( n = 3 ) from three independent repeats with one - way ANOVA analysis and Tukey test ( p < 0 . 05 ) . Different letters indicate significant differences . ( D ) flg22 - induced callose deposition in transgenic lines . Leaves of four - week - old plants were inoculated with 500 nM flg22 , and callose deposits were stained with aniline blue at 20 hpi . Number of callose deposits was quantified with Image J ( mean ± SE , n = 10 ) . All experiments were repeated three times with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Phenotype of plants complemented with WT DDL or DDL12E1. Four-week-old plants of two independent transgenic lines of pDDL::DDL-FLAG (W7 and W18) or pDDL::DDL12E1-FLAG(E11 and E15) were photographed. Bar=1cm.(A) Phenotype of plants complemented with WT DDL or DDL12E1. Four-week-old plants of two independent transgenic lines of pDDL::DDL-FLAG (W7 and W18) or pDDL::DDL12E1-FLAG(E11 and E15). The expression of transgene was determined by an α-FLAGimmunoblot (bottom).(B) Susceptibility of complementation lines to Pst DC3000 infections. Four-week-old transgenic plants were inoculated with Pst DC3000 (OD600=5x10-4) and the bacterial number was determined at 0 and 3 dpi.(C) PR1 and PR5 induction in transgenic lines. Leaves of four-week-old plants were inoculated with Pst DC3000 (OD600=0.01), and collected at 0, and 24 hpi for qRT-PCR analysis. The data in B and C are shown as mean ± SD (n=3) from three independent repeats with one-way ANOVA analysis and Tukey test (p<0.05). Different letters indicate significant differences.(D) flg22-induced callose deposition in transgenic lines. Leaves of four-week-old plants were inoculated with 500 nM flg22, and callose deposits were stained with aniline blue at 20 hpi. Number of callose deposits was quantified with Image J (mean ± SE, n=10). All experiments were repeated three times with similar results.


Figure 1 ( G ) Reduced NMDA / AMPA ratio at SC - CA1 synapses of PtenΔC / ΔC mice ( P17 - 21 ) . The NMDAR component was obtained 60 ms after stimulation . ( ratio , n = 11 cells from 8 mice for WT and 8 ( 8 ) for ΔC ; decay , n = 12 cells from 8 mice for WT and 11 ( 8 ) for ΔC , * * P < 0 . 01 , ns , not significant , Mann - Whitney U test ) . The error bars represent SEM . ( H ) Reduced amplitude but not frequency of NMDA - mEPSCs at PtenΔC / ΔC CA1 pyramidal neurons . ( n = 17 cells from 4 mice for WT and 18 ( 4 ) for ΔC , * * P < 0 . 01 , ns , not significant , Mann - Whitney U test , Student ' s t - test ) . The error bars represent SEM . ( I ) Reduced NMDA / AMPA ratio at synapses in layer II / III pyramidal neurons in the prelimbic region of the mPFC of PtenΔC / ΔC mice ( P20 - 21 ) . The NMDAR component was obtained 60 ms after stimulation . ( n = 9 cells from 3 mice for WT and 10 ( 3 ) for ΔC , * P < 0 . 05 , * * P < 0 . 01 , Student ' s t - test , Mann - Whitney U test ) . The error bars represent SEM . Data information : ( C - F ) Results from the last 10 - minute recordings are shown in scatter plots .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(G) Reduced NMDA/AMPA ratio at SC-CA1 synapses of PtenΔC/ΔC mice (P17-21). The NMDAR component was obtained 60 ms after stimulation. (ratio, n = 11 cells from 8 mice for WT and 8 (8) for ΔC; decay, n = 12 cells from 8 mice for WT and 11 (8) for ΔC, **P < 0.01, ns, not significant, Mann-Whitney U test). The error bars represent SEM. (H) Reduced amplitude but not frequency of NMDA -mEPSCs at PtenΔC/ΔC CA1 pyramidal neurons. (n = 17 cells from 4 mice for WT and 18 (4) for ΔC, **P < 0.01, ns, not significant, Mann-Whitney U test, Student's t-test). The error bars represent SEM. (I) Reduced NMDA/AMPA ratio at synapses in layer II/III pyramidal neurons in the prelimbic region of the mPFC of PtenΔC/ΔC mice (P20-21). The NMDAR component was obtained 60 ms after stimulation. (n = 9 cells from 3 mice for WT and 10 (3) for ΔC, *P < 0.05, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM. Data information: (C-F) Results from the last 10-minute recordings are shown in scatter plots.


Figure 2A ) A volcano plot showing DEGs ( adjusted p value < 0 . 1 ) derived from RNA - Seq results for PtenΔC / ΔC and WT mice ( P21 ) . For the calculation of adjusted p value , we have employed the R package DESeq2 where the P ‐ values obtained by the Wald test are corrected for multiple testing using the Benjamini and Hochberg method . ( n = 3 mice for WT and ΔC ) . See also Dataset EV1 for the full RNA - Seq results . C ) Immunoblot validation of the increase in SLC6A20 protein levels in hippocampal lysates of PtenΔC / ΔC mice ( P21 ) . We denoted the antibody as ' SLC6A20 ' here , not ' SLC6A20A ' , because this antibody was generated using a synthetic peptide sequence ( YNEPSNNCQKHAI ) that is commonly present in SLC6A20A and SLC6A20B , being a pan - SLC6A20 antibody ( ThermoFisher ) . ( n = 3 mice for WT and ∆ C , * P < 0 . 05 , Student ' s t - test ) . The error bars represent SEM . ( D , E ) Increased phosphorylation of β - catenin at Ser - 675 but not at Ser - 552 in the nucleus - enriched P1 fraction but not in whole - brain lysates of PtenΔC / ΔC mice ( 3 months ) . Note that total levels of β - catenin were not changed in the P1 fraction or whole - brain lysates . ( n = 4 mice for WT - WB / P1 and ΔC - WB / P1 , * P < 0 . 05 , ns , not significant , Student ' s t - test ) . The error bars represent SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) A volcano plot showing DEGs (adjusted p value < 0.1) derived from RNA-Seq results for PtenΔC/ΔC and WT mice (P21). For the calculation of adjusted p value, we have employed the R package DESeq2 where the P‐values obtained by the Wald test are corrected for multiple testing using the Benjamini and Hochberg method. (n = 3 mice for WT and ΔC). See also Dataset EV1 for the full RNA-Seq results.C) Immunoblot validation of the increase in SLC6A20 protein levels in hippocampal lysates of PtenΔC/ΔC mice (P21). We denoted the antibody as 'SLC6A20' here, not 'SLC6A20A', because this antibody was generated using a synthetic peptide sequence (YNEPSNNCQKHAI) that is commonly present in SLC6A20A and SLC6A20B, being a pan-SLC6A20 antibody (ThermoFisher). (n = 3 mice for WT and ∆C, *P < 0.05, Student's t-test). The error bars represent SEM.(D,E) Increased phosphorylation of β-catenin at Ser-675 but not at Ser-552 in the nucleus-enriched P1 fraction but not in whole-brain lysates of PtenΔC/ΔC mice (3 months). Note that total levels of β-catenin were not changed in the P1 fraction or whole-brain lysates. (n = 4 mice for WT-WB/P1 and ΔC-WB/P1, *P < 0.05, ns, not significant, Student's t-test). The error bars represent SEM.


Figure 3 ( B , C ) Decreased extracellular levels of proline and glycine in the brains of PtenΔC / ΔC mice ( 2 - 4 month ) , as determined by microdialysis in the hippocampus ( see Materials and Methods for details ) . ( proline , n = 13 mice for WT and 8 for ΔC , * * P < 0 . 01 , Student ' s t - test ; glycine , n = 9 for WT and 5 for ∆ C , * * P < 0 . 01 , Student ' s t - test , Mann - Whitney U test ) . The error bars represent SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B,C) Decreased extracellular levels of proline and glycine in the brains of PtenΔC/ΔC mice (2-4 month), as determined by microdialysis in the hippocampus (see Materials and Methods for details). (proline, n = 13 mice for WT and 8 for ΔC, **P < 0.01, Student's t-test; glycine, n = 9 for WT and 5 for ∆C, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM.


Figure 4 ( B ) Normalization of NMDAR - dependent HFS - LTP at synapses of PtenΔC / ΔC mice ( 4 - 5 weeks ) by DCS treatment ( 20 μM ) , as shown by fEPSP slopes . ( n = 15 slices from 9 mice for WT - V / vehicle , 11 ( 5 ) for WT - D / DCS , 16 ( 10 ) for ΔC - V , 13 ( 7 ) for ΔC - D , * * P < 0 . 01 , ns , not significant , two - way ANOVA with Tukey ' s test ) . The grey traces represent the baseline fEPSP prior to LTP induction . The error bars represent SEM . ( F ) Normalization of NMDAR - dependent HFS - LTP at synapses of PtenΔC / ΔC mice ( 4 - 5 weeks ) by sarcosine treatment ( 750 µM ) , as shown by fEPSP slopes . Note that the data for WT - V and ΔC - V are identical to those shown in Fig . 4B because the whole experiments were performed together ; we generated independent figures for DCS and sarcosine results for the clear presentation of the data . ( n = 15 slices from 9 mice for WT - V / vehicle , 9 ( 4 ) for WT - Sarc / Sarcosine , 16 ( 10 ) for ΔC - V / Vehicle , 18 ( 4 ) for ΔC - S , * P < 0 . 05 , * * * P < 0 . 001 , ns , not significant , two - way ANOVA with Tukey ' s test ) . The grey traces represent the baseline fEPSP prior to LTP induction . The error bars represent SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) Normalization of NMDAR-dependent HFS-LTP at synapses of PtenΔC/ΔC mice (4-5 weeks) by DCS treatment (20 μM), as shown by fEPSP slopes. (n = 15 slices from 9 mice for WT-V/vehicle, 11 (5) for WT-D/DCS, 16 (10) for ΔC-V, 13 (7) for ΔC-D, **P < 0.01, ns, not significant, two-way ANOVA with Tukey's test). The grey traces represent the baseline fEPSP prior to LTP induction. The error bars represent SEM.(F) Normalization of NMDAR-dependent HFS-LTP at synapses of PtenΔC/ΔC mice (4-5 weeks) by sarcosine treatment (750 µM), as shown by fEPSP slopes. Note that the data for WT-V and ΔC-V are identical to those shown in Fig. 4B because the whole experiments were performed together; we generated independent figures for DCS and sarcosine results for the clear presentation of the data. (n = 15 slices from 9 mice for WT-V/vehicle, 9 (4) for WT-Sarc/Sarcosine, 16 (10) for ΔC-V/Vehicle, 18 (4) for ΔC-S, *P < 0.05, ***P < 0.001, ns, not significant, two-way ANOVA with Tukey's test). The grey traces represent the baseline fEPSP prior to LTP induction. The error bars represent SEM.


Figure 5 ( D ) PtenΔC / ΔC mice ( 2 - 4 months ) treated with Slc6a20a - ASO display partially normalized levels of whole - brain glycine , as shown by the lack of difference between saline - treated WT and ASO - treated PtenΔC / ΔC mice . Note that the glycine levels observed here are ~ 4 times lower than those measured in Fig . 3A , which could be attributable to different mouse ages ( P21 in Fig . 3A vs . 2 - 4 months in Fig . 5D ) , absence and presence of ASO injection , or lot - to - lot variation of the ELISA kits . ( n = 8 mice for WT - saline , 5 for WT - ASO , 7 for ΔC - saline , and 8 for ΔC - ASO , * * P < 0 . 01 , ns , not significant , two - way ANOVA with Tukey ' s test ) . The error bars represent SEM . ( E ) PtenΔC / ΔC mice ( P20 - 37 ) treated with Slc6a20a - ASO display an NMDA / AMPA ratio at hippocampal SC - CA1 synapses that is comparable to that in ASO - treated WT mice . ( n = 11 neurons from 5 mice for WT - ASO , 10 ( 4 ) for ΔC - ASO , ns , not significant , Mann - Whitney ' s U test ) . The error bars represent SEM . ( G ) PtenΔC / ΔC mice ( 2 - 4 months ) treated with Slc6a20a - ASO display normal levels of climbing in the Laboras test . n = 6 mice for WT - saline , 7 for WT - ASO , 7 for ΔC - saline , and 8 for ΔC - ASO , * P < 0 . 05 , * * P < 0 . 01 , ns , not significant , two - way ANOVA with Bonferroni ' s test . The error bars represent SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(D) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display partially normalized levels of whole-brain glycine, as shown by the lack of difference between saline-treated WT and ASO-treated PtenΔC/ΔC mice. Note that the glycine levels observed here are ~4 times lower than those measured in Fig. 3A, which could be attributable to different mouse ages (P21 in Fig. 3A vs. 2-4 months in Fig. 5D), absence and presence of ASO injection, or lot-to-lot variation of the ELISA kits. (n = 8 mice for WT-saline, 5 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, **P < 0.01, ns, not significant, two-way ANOVA with Tukey's test). The error bars represent SEM.(E) PtenΔC/ΔC mice (P20-37) treated with Slc6a20a-ASO display an NMDA/AMPA ratio at hippocampal SC-CA1 synapses that is comparable to that in ASO-treated WT mice. (n = 11 neurons from 5 mice for WT-ASO, 10 (4) for ΔC-ASO, ns, not significant, Mann-Whitney's U test). The error bars represent SEM.(G) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display normal levels of climbing in the Laboras test. n = 6 mice for WT-saline, 7 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, *P < 0.05, **P <0.01, ns, not significant, two-way ANOVA with Bonferroni's test. The error bars represent SEM.


Figure 6 ( D ) Increased extracellular levels of glycine and proline in the brain of Slc6a20a + / - and Slc6a20a - / - mice , as shown by microdialysis analyses . Note that glycine levels are increased in both Slc6a20a + / - and Slc6a20a - / - mice , whereas proline levels are increased only in Slc6a20a - / - mice . ( glycine , n = 19 mice for WT , 11 for HT , 8 for KO , * * P < 0 . 01 , Mann - Whitney U test ; proline , n = 15 mice for WT , 11 for HT , 14 for KO , * * * P < 0 . 001 , ns , not significant , Mann - Whitney U test ) . The error bars represent SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(D) Increased extracellular levels of glycine and proline in the brain of Slc6a20a+/- and Slc6a20a-/- mice, as shown by microdialysis analyses. Note that glycine levels are increased in both Slc6a20a+/- and Slc6a20a-/- mice, whereas proline levels are increased only in Slc6a20a-/- mice. (glycine, n = 19 mice for WT, 11 for HT, 8 for KO, **P < 0.01, Mann-Whitney U test; proline, n = 15 mice for WT, 11 for HT, 14 for KO, ***P < 0.001, ns, not significant, Mann-Whitney U test). The error bars represent SEM.


Figure 7 ( B ) Glycine / proline - induced currents for mouse SLC6A20A are dependent on sodium chloride , as shown by the suppression of the currents when sodium chloride is replaced with choline chloride or sodium gluconate . Each experimental condition was sequentially applied to HEK293T cells , as shown in Appendix Fig . S7B . The indicated currents are average values from an ensemble of multiple ( ~ 20 ) cells in a single well . ( glycine , n = 21 cells for untransfected / blank , 17 for human SLC6A20 - V1 , 25 for human SLC6A20 - V2 , 33 for mSLC6A20A , 28 for mSLC6A20B * * * P < 0 . 001 ( relative to buffer not containing glycine ) , two - way ANOVA with Bonferroni ' s test ; proline , n = 17 cells for untransfected , 23 for human SLC6A20 - V1 , 47 for SLC6A20 - V2 , 31 for mSLC6A20A , and 31 for mSLC6A20B , * * * P < 0 . 001 , two - way ANOVA with Bonferroni ' s test ) . The error bars represent SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B) Glycine/proline-induced currents for mouse SLC6A20A are dependent on sodium chloride, as shown by the suppression of the currents when sodium chloride is replaced with choline chloride or sodium gluconate. Each experimental condition was sequentially applied to HEK293T cells, as shown in Appendix Fig. S7B. The indicated currents are average values from an ensemble of multiple (~20) cells in a single well. (glycine, n = 21 cells for untransfected/blank, 17 for human SLC6A20-V1, 25 for human SLC6A20-V2, 33 for mSLC6A20A, 28 for mSLC6A20B ***P < 0.001 (relative to buffer not containing glycine), two-way ANOVA with Bonferroni's test; proline, n = 17 cells for untransfected, 23 for human SLC6A20-V1, 47 for SLC6A20-V2, 31 for mSLC6A20A, and 31 for mSLC6A20B, ***P < 0.001, two-way ANOVA with Bonferroni's test). The error bars represent SEM.


Figure 1 ( A ) EM analysis of cortical synapses derived from WT and MPS - IIIA mice at different ages . The size of synaptic vesicles was quantified from 400 - 500 vesicles ( taken from 5 mice for each genotype at each time point ) and expressed as the average of vesicle diameter ( nm ) . The number of synaptic vesicles per synapse was quantified from 20 different images ( taken from 5 mice for each genotype at each time point ) , normalized by the length of synaptic cleft and expressed as % of WT . The synaptic density was measured from 20 different images ( taken from 5 mice for each genotype at each time point ) as the number of synapses / area ( # / 500μm2 ) and expressed as % of WT . Arrows indicate the synaptic cleft , while asterisks indicate abnormal vacuoles ( B , C ) The size of the lysosomal compartment was evaluated by WB ( B ) analysis in the brain of MPS - IIIA mice at indicated ages . Quantitation of WB by densitometry analysis ( ImageJ ) was shown ( B ) . ( B , C ) The size of the lysosomal compartment was evaluated by IF ( C ) analysis in the brain of MPS - IIIA mice at indicated ages . ( D ) Extracellular recordings ( fEPSPs ) in hippocampal brain slices from 6 months - old WT and MPS - IIIA mice . The left panel shows representative fEPSP traces . Summary graphs show the fEPSPs slope as a function of either the applied stimulus intensity or the prevolley amplitude . Data are the average of the values from 9 slices ( from 9 mice ) in the WT group and 6 slices ( from 5 mice ) in the MPS - IIIA . * p < 0 . 05 , repeated measure ANOVA .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) EM analysis of cortical synapses derived from WT and MPS-IIIA mice at different ages. The size of synaptic vesicles was quantified from 400-500 vesicles (taken from 5 mice for each genotype at each time point) and expressed as the average of vesicle diameter (nm). The number of synaptic vesicles per synapse was quantified from 20 different images (taken from 5 mice for each genotype at each time point), normalized by the length of synaptic cleft and expressed as % of WT. The synaptic density was measured from 20 different images (taken from 5 mice for each genotype at each time point) as the number of synapses/area(#/500μm2) and expressed as % of WT.Arrows indicate the synaptic cleft, while asterisks indicate abnormal vacuoles(B, C) The size of the lysosomal compartment was evaluated by WB (B) analysis in the brain of MPS-IIIA mice at indicated ages. Quantitation of WB by densitometry analysis (ImageJ) was shown (B).(B, C) The size of the lysosomal compartment was evaluated by IF (C) analysis in the brain of MPS-IIIA mice at indicated ages.(D) Extracellular recordings (fEPSPs) in hippocampal brain slices from 6 months-old WT and MPS-IIIA mice. The left panel shows representative fEPSP traces. Summary graphs show the fEPSPs slope as a function of either the applied stimulus intensity or the prevolley amplitude. Data are the average of the values from 9 slices (from 9 mice) in the WT group and 6 slices (from 5 mice) in the MPS-IIIA. *p<0.05, repeated measure ANOVA.


Figure 2 ( A ) Synaptic terminal endocytosis was analyzed in WT and MPS - IIIA DIV14 hippocampal neurons by quantification of incorporated FM1 - 43 dye fluorescence in ~ 300 individual boutons ( taken from 4 - 5 coverslips for each group ) . Fluorescence intensities were expressed as arbitrary units ( A . F . U . ) and displayed both as a distribution and as mean values ± s . e . m . ( B ) WT and MPS - IIIA hippocampal neurons ( DIV14 ) were transfected with v - Glut1 - pHluorin - mCherry plasmid and synaptic recycling was evaluated by the fluorescence change of the probe from 80 - 100 individual boutons ( taken from 4 - 5 coverslips for each group ) . Fluorescence intensity was quantified at each bouton during KCl perfusion , normalized to the fluorescence value obtained upon rapid alkalization ( NH4Cl perfusion ) and expressed as ∆ fluorescence ( normalized to baseline ; ΔFKCl / ΔF NH4Cl ) . Values were displayed both as fluorescence traces ( means ± s . e . m . at each time frame ) and maximum fluorescence after KCl perfusion ( means ± s . e . m . ) . Representative panels on the right show the fluorescence intensity change in control neurons ( WT ) . Pseudo - colour was applied to better reveal fluorescence changes . Note that NH4Cl alkalinizes all vesicles revealing the total ( recycling + resting ) pool in neuronal cells analyzed . ( C ) WT hippocampal neurons at DIV10 were treated with a lysosome inhibitor ' s cocktail for 3 days . The size of lysosomal compartment was evaluated in both treated and control untreated WT neurons by EM examination . Quantitation analysis was performed on 30 different images . ( D , E ) Endocytosis ( D ) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in ( A , B ) . ( D , E ) exocytosis ( E ) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in ( A , B ) . ( F ) EM analysis of synaptic terminals was performed in treated and control untreated WT neurons . The number of synaptic vesicles per synapse was quantified from ~ 20 different images ( taken from 3 ultrathin sections for each group ) , normalized by the length of synaptic cleft and expressed as % of WT . Arrows indicate the synaptic cleft .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Synaptic terminal endocytosis was analyzed in WT and MPS-IIIA DIV14 hippocampal neurons by quantification of incorporated FM1-43 dye fluorescence in ~300 individual boutons (taken from 4-5 coverslips for each group). Fluorescence intensities were expressed as arbitrary units (A.F.U.) and displayed both as a distribution and as mean values ± s.e.m.(B) WT and MPS-IIIA hippocampal neurons (DIV14) were transfected with v-Glut1-pHluorin-mCherry plasmid and synaptic recycling was evaluated by the fluorescence change of the probe from 80-100 individual boutons (taken from 4-5 coverslips for each group). Fluorescence intensity was quantified at each bouton during KCl perfusion, normalized to the fluorescence value obtained upon rapid alkalization (NH4Cl perfusion) and expressed as ∆fluorescence (normalized to baseline; ΔFKCl/ΔF NH4Cl). Values were displayed both as fluorescence traces (means ± s.e.m. at each time frame) and maximum fluorescence after KCl perfusion (means ± s.e.m.). Representative panels on the right show the fluorescence intensity change in control neurons (WT). Pseudo-colour was applied to better reveal fluorescence changes.Note that NH4Cl alkalinizes all vesicles revealing the total (recycling + resting) pool in neuronal cells analyzed.(C) WT hippocampal neurons at DIV10 were treated with a lysosome inhibitor's cocktail for 3 days. The size of lysosomal compartment was evaluated in both treated and control untreated WT neurons by EM examination. Quantitation analysis was performed on 30 different images.(D, E) Endocytosis (D) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in (A, B).(D, E) exocytosis (E) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in (A, B).(F) EM analysis of synaptic terminals was performed in treated and control untreated WT neurons. The number of synaptic vesicles per synapse was quantified from ~20 different images (taken from 3 ultrathin sections for each group), normalized by the length of synaptic cleft and expressed as % of WT. Arrows indicate the synaptic cleft.


Figure 3 ( A ) VAMP2 , SNAP - 25 and Syntaxin - 1 SNAREs were immunoblotted in WT and MPS - IIIA synaptosomal brain samples at indicated ages . Synapsin - 1 was also blotted as a control . Quantitation of WB was shown . ( B ) Confocal microscopy images of WT and MPS - IIIA hippocampal neurons ( DIV14 ) double labeled with anti - synapsin - 1 ( presynaptic marker ; red ) and either anti - VAMP2 or anti - SNAP25 antibodies ( green ) . The merges ( yellow ) of confocal images are shown . SNAP25 - synapsin - 1 and VAMP2 - synapsin - 1 co - localizations were quantified using the Manders ' Colocalization Coefficients ( MCC ) ( ImageJ ) and displayed as % ( MCC x 100 ) of Synapsin - 1 co - localizing with either SNAP25 or VAMP2 ( means ± s . e . m . from 15 different images taken from 4 - 5 coverslips for each group ) . ( C ) VAMP2 and SNAP - 25 protein levels were quantified by immunoblot analysis in WT and MPS - IIIA hippocampal neurons ( DIV14 ) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 ( 100 % ) . The proteasome was inhibited as indicated . ( D ) SDS - resistant complex levels were evaluated in WT and MPS - IIIA total brain samples at indicated ages by immunoblotting of non - boiled samples with VAMP2 or Syntaxin - 1 antibodies . Quantitation of WB was shown . ( E ) Total brain lysates were immunoprecipitated with antibodies to Syntaxin - 1 and co - immunoprecipitated VAMP2 and SNAP25 proteins were revealed by WB analysis . The levels of immunoprecipitated proteins were quantified . ( F ) α - Synuclein and CSPα were immunoblotted in WT and MPS - IIIA total brain lysates at indicated ages Total protein levels were quantified . ( G ) α - Synuclein and CSPα were immunoblotted in WT and MPS - IIIA synaptosomal fractions at indicated ages . Hsc70 , an heat shock cognate protein , which forms together with CSPα and SGT ( small glutamine - rich tetratricopeptide repeat domain protein ) the " chaperon machine " was also blotted . Protein levels were quantified .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) VAMP2, SNAP-25 and Syntaxin-1 SNAREs were immunoblotted in WT and MPS-IIIA synaptosomal brain samples at indicated ages. Synapsin-1 was also blotted as a control. Quantitation of WB was shown.(B) Confocal microscopy images of WT and MPS-IIIA hippocampal neurons (DIV14) double labeled with anti-synapsin-1 (presynaptic marker; red) and either anti-VAMP2 or anti-SNAP25 antibodies (green). The merges (yellow) of confocal images are shown. SNAP25-synapsin-1 and VAMP2-synapsin-1 co-localizations were quantified using the Manders' Colocalization Coefficients (MCC) (ImageJ) and displayed as % (MCC x 100) of Synapsin-1 co-localizing with either SNAP25 or VAMP2 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group).(C) VAMP2 and SNAP-25 protein levels were quantified by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 (100 %). The proteasome was inhibited as indicated.(D) SDS-resistant complex levels were evaluated in WT and MPS-IIIA total brain samples at indicated ages by immunoblotting of non-boiled samples with VAMP2 or Syntaxin-1 antibodies. Quantitation of WB was shown.(E) Total brain lysates were immunoprecipitated with antibodies to Syntaxin-1 and co-immunoprecipitated VAMP2 and SNAP25 proteins were revealed by WB analysis. The levels of immunoprecipitated proteins were quantified.(F) α-Synuclein and CSPα were immunoblotted in WT and MPS-IIIA total brain lysates at indicated ages Total protein levels were quantified.(G) α-Synuclein and CSPα were immunoblotted in WT and MPS-IIIA synaptosomal fractions at indicated ages. Hsc70, an heat shock cognate protein, which forms together with CSPα and SGT (small glutamine-rich tetratricopeptide repeat domain protein) the "chaperon machine" was also blotted. Protein levels were quantified.


Figure 4 ( A ) WB analysis of LC3 and p62 ( an autophagy substrate ) was performed on WT and MPS - IIIA brain samples at indicated ages . WB quantitation was shown . ( B ) α - Synuclein was immunoblotted in WT and MPS - IIIA both total and synaptosomal brain fractions at indicated ages after sequential extraction with detergents with increased strength . Soluble ( Sol . ) , lowly insoluble ( L . Insol . ) and highly insoluble ( H . Insol . ) forms correspond to the protein solubilized respectively in Triton - X100 , SDS 10 % and 8M UREA . ( C ) Co - immunofluorescence analysis of α - synuclein with SMI - 32 in WT and MPS - IIIA hippocampal neurons ( DIV14 ) . α - Synuclein synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image . Quantification of α - synuclein synaptic puncta was calculated from 30 different enlarged images c . ( D ) Confocal analysis of α - synuclein ( green ) and LAMP1 ( red ) in WT and MPS - IIIA hippocampal neurons ( DIV14 ) . Enlarged merge images are also shown . Co - localization was quantified using the MCC coefficient ( ImageJ ) and displayed as % ( MCC x 100 ) of α - synuclein co - localizing with LAMP1 ( means ± s . e . m . from 15 different images taken from 4 - 5 coverslips for each group ) . ( E ) CSPα was immunoblotted in WT and MPS - IIIA total brain lysates at indicated ages after sequential extraction with detergents with increased strength as in ( B ) . ( F ) Co - immunofluorescence analysis of CSPα and SMI - 32 in DIV14 hippocampal neurons . CSPα - synaptic puncta were quantified as in ( C ) . ( G ) CSPα protein levels were evaluated by immunoblot analysis in WT and MPS - IIIA hippocampal neurons ( DIV14 ) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 ( 100 % ) . The proteasome was inhibited as indicated . ( H ) Palmitoylation - dependent shift in the molecular weight of CSPα was evaluated in WT and MPS - IIIA brain samples at indicated ages by immunoblotting CSPα in boiled samples prepared without exposure to sulphydryl agents ( beta - mercaptoethanol or dithiothreitol ) . ( I ) The protein levels of RPN10 ( the regulatory subunit of 26S proteasome ) and ubiquitinated proteins formed by Lys - 48 ( K48 ) residue linkage ( involved in protein degradation via the proteasome ) were evaluated in WT and MPS - IIIA brain samples at indicated ages by WB analysis . WB quantitation was shown . ( L ) Proteasome activity was evaluated by measuring the chymotrypsin - like activity in WT and MPS - IIIA mouse brain samples at different ages . Proteasome activity was expressed as % of WT

O O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) WB analysis of LC3 and p62 (an autophagy substrate) was performed on WT and MPS-IIIA brain samples at indicated ages. WB quantitation was shown.(B) α-Synuclein was immunoblotted in WT and MPS-IIIA both total and synaptosomal brain fractions at indicated ages after sequential extraction with detergents with increased strength. Soluble (Sol.), lowly insoluble (L. Insol.) and highly insoluble (H. Insol.) forms correspond to the protein solubilized respectively in Triton-X100, SDS 10% and 8M UREA.(C) Co-immunofluorescence analysis of α-synuclein with SMI-32 in WT and MPS-IIIA hippocampal neurons (DIV14). α-Synuclein synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image. Quantification of α-synuclein synaptic puncta was calculated from 30 different enlarged images c.(D) Confocal analysis of α-synuclein (green) and LAMP1 (red) in WT and MPS-IIIA hippocampal neurons (DIV14). Enlarged merge images are also shown. Co-localization was quantified using the MCC coefficient (ImageJ) and displayed as % (MCC x 100) of α-synuclein co-localizing with LAMP1 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group).(E) CSPα was immunoblotted in WT and MPS-IIIA total brain lysates at indicated ages after sequential extraction with detergents with increased strength as in (B).(F) Co-immunofluorescence analysis of CSPα and SMI-32 in DIV14 hippocampal neurons. CSPα- synaptic puncta were quantified as in (C).(G) CSPα protein levels were evaluated by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 (100 %). The proteasome was inhibited as indicated.(H) Palmitoylation-dependent shift in the molecular weight of CSPα was evaluated in WT and MPS-IIIA brain samples at indicated ages by immunoblotting CSPα in boiled samples prepared without exposure to sulphydryl agents (beta-mercaptoethanol or dithiothreitol).(I) The protein levels of RPN10 (the regulatory subunit of 26S proteasome) and ubiquitinated proteins formed by Lys-48 (K48) residue linkage (involved in protein degradation via the proteasome) were evaluated in WT and MPS-IIIA brain samples at indicated ages by WB analysis. WB quantitation was shown.(L) Proteasome activity was evaluated by measuring the chymotrypsin-like activity in WT and MPS-IIIA mouse brain samples at different ages. Proteasome activity was expressed as % of WT activity


Figure 5 ( A ) Autophagy was monitored in WT hippocampal neurons treated with the lysosome inhibitor ' s cocktail for 3 days by immunoblot analysis with anti - LC3 antibodies . As control DIV10 WT neurons were left untreated and monitored by LC3 blot over the three days period . The LC3 - II levels quantitation was shown . ( B ) Proteasome activity was measured in treated and control untreated WT neurons using pZsProSensor - 1 vector ( green ) . Cells were co - stained with anti SMI - 32 ( blu ) . Proteasome activity was quantified by measuring the green fluorescence ( inversely correlated with proteasome activity ) in 10 different cells ( taken from 4 - 5 coverslips for each group ) and expressed as fold to WT . ( C ) Activation of calpain 2 and caspase - 3 proteolytic systems was evaluated by WB measurement of the protein levels of calpain 2 and caspase - 3 ( both full - length and activated cleaved forms of ~ 17 KDa ) in treated and control untreated WT neurons . Arrow indicated the full - length caspase - 3 protein . ( D ) After 3 days - treatment cell lysates from treated and control untreated WT neurons were immunoblotted with α - synuclein and CSPα and protein levels were quantified . ( E ) Treated and control untreated WT neurons were subjected to α - synuclein ( green ) and LAMP1 ( red ) confocal analysis . Enlarged merge images are also shown . Co - localization was quantified using the MCC coefficient ( ImageJ ) and displayed as % ( MCC x 100 ) of α - synuclein co - localizing with LAMP1 ( means ± s . e . m . from 15 different images taken from 4 - 5 coverslips for each group ) . Cells were also co - stained with anti SMI - 32 . ( F ) Treated and control untreated WT neurons were subjected to CSPα / SMI - 32 co - immunofluorescence . CSPα synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image . Quantification of CSPα synaptic puncta was calculated from 30 different enlarged images taken from 4 - 5 coverslips for each group . ( G ) SDS - resistant complex levels in treated and control untreated WT neurons were evaluated by immunoblotting analysis of non - boiled samples with VAMP2 or SNAP - 25 antibodies . The amounts of SNARE complexes were quantified .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Autophagy was monitored in WT hippocampal neurons treated with the lysosome inhibitor's cocktail for 3 days by immunoblot analysis with anti-LC3 antibodies. As control DIV10 WT neurons were left untreated and monitored by LC3 blot over the three days period. The LC3-II levels quantitation was shown.(B) Proteasome activity was measured in treated and control untreated WT neurons using pZsProSensor-1 vector (green). Cells were co-stained with anti SMI-32 (blu). Proteasome activity was quantified by measuring the green fluorescence (inversely correlated with proteasome activity) in 10 different cells (taken from 4-5 coverslips for each group) and expressed as fold to WT.(C) Activation of calpain 2 and caspase-3 proteolytic systems was evaluated by WB measurement of the protein levels of calpain 2 and caspase-3 (both full-length and activated cleaved forms of ~17 KDa) in treated and control untreated WT neurons. Arrow indicated the full-length caspase-3 protein.(D) After 3 days-treatment cell lysates from treated and control untreated WT neurons were immunoblotted with α-synuclein and CSPα and protein levels were quantified.(E) Treated and control untreated WT neurons were subjected to α-synuclein (green) and LAMP1 (red) confocal analysis. Enlarged merge images are also shown. Co-localization was quantified using the MCC coefficient (ImageJ) and displayed as % (MCC x 100) of α-synuclein co-localizing with LAMP1 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group). Cells were also co-stained with anti SMI-32.(F) Treated and control untreated WT neurons were subjected to CSPα/SMI-32 co-immunofluorescence. CSPα synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image. Quantification of CSPα synaptic puncta was calculated from 30 different enlarged images taken from 4-5 coverslips for each group.(G) SDS-resistant complex levels in treated and control untreated WT neurons were evaluated by immunoblotting analysis of non-boiled samples with VAMP2 or SNAP-25 antibodies. The amounts of SNARE complexes were quantified.


Figure 6 ( A ) α - Synuclein , CSPα , VAMP2 and SNAP - 25 were immunoblotted in total brain homogenates and / or synaptosomal fractions of 1 . 5 month - old WT ( CSPα + / + / α - syn + / + ) , CSPα - / - , CSPα + / - , α - syn - / - , α - syn + / - and CSPα + / - / α - syn + / - . Synapsin - 1 was also blotted as a control . Quantitation of WB in synaptosomal samples was shown . ( B ) SDS - resistant complex levels were evaluated in brain homogenates derived from mice with indicated genotypes by immunoblotting of non - boiled samples with VAMP2 or Syntaxin - 1 antibodies . Quantitation of WB was shown . ( C ) EM analysis of cortical synapses derived from mice with indicated genotypes . The number of synaptic vesicles per synapse was quantified from 40 different images ( taken from 5 different mice for each genotype ) , normalized by the length of synaptic cleft and expressed as % of WT . The size of synaptic vesicles was quantified from 400 - 500 vesicles ( taken from 5 different mice for each genotype ) and expressed as the average of vesicle diameter ( nm ) Arrows indicate the synaptic cleft . ( D ) Extracellular recordings ( fEPSPs ) in hippocampal brain slices from 1 . 5 months - old WT ( CSPα + / + / α - syn + / + ) and CSPα + / - / α - syn + / - mice . The left panel shows representative fEPSP traces . Summary graphs show the fEPSPs slope as a function of either the applied stimulus intensity or the prevolley amplitude . Data are the average of the values from 5 slices ( from 4 mice ) in the WT group and 5 slices ( from 4 mice ) in the CSPα + / - / α - syn + / - . * p < 0 . 05 , repeated measure ANOVA .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) α-Synuclein, CSPα, VAMP2 and SNAP-25 were immunoblotted in total brain homogenates and/or synaptosomal fractions of 1.5 month-old WT (CSPα+/+/α-syn+/+), CSPα-/-, CSPα+/-, α-syn-/-, α-syn+/- and CSPα+/-/α-syn+/-. Synapsin-1 was also blotted as a control. Quantitation of WB in synaptosomal samples was shown.(B) SDS-resistant complex levels were evaluated in brain homogenates derived from mice with indicated genotypes by immunoblotting of non-boiled samples with VAMP2 or Syntaxin-1 antibodies. Quantitation of WB was shown.(C) EM analysis of cortical synapses derived from mice with indicated genotypes. The number of synaptic vesicles per synapse was quantified from 40 different images (taken from 5 different mice for each genotype), normalized by the length of synaptic cleft and expressed as % of WT. The size of synaptic vesicles was quantified from 400-500 vesicles (taken from 5 different mice for each genotype) and expressed as the average of vesicle diameter (nm) Arrows indicate the synaptic cleft.(D) Extracellular recordings (fEPSPs) in hippocampal brain slices from 1.5 months-old WT (CSPα+/+/α-syn+/+) and CSPα+/-/α-syn+/- mice. The left panel shows representative fEPSP traces. Summary graphs show the fEPSPs slope as a function of either the applied stimulus intensity or the prevolley amplitude. Data are the average of the values from 5 slices (from 4 mice) in the WT group and 5 slices (from 4 mice) in the CSPα+/-/α-syn+/-. *p<0.05, repeated measure ANOVA.


Figure 7 ( A ) Anti - myc immunostaining in different brain regions derived from 10 - months - old MPS - IIIA mice intraventricularly injected with AAV2 / 9 vectors encoding CSPα under Synapsin - 1 promoter ( MPS - IIIA - myc - CSPα experimental group ) . As a control immunostaining was performed in brain sections from 10 - months - old MPS - IIIA mice injected with empty AAV2 / 9 vectors ( MPS - IIIA - empty experimental groups ) . To evaluate co - localization between exogenous CSPα and presynaptic compartment brain sections were also co - stained with anti - myc and anti - synapsin1 antibodies ( confocal images are also shown ) . ( B ) Anti - myc WB in synaptosomal fractions derived from MPS - IIIA - myc - CSPα mice . As a control WB was performed in samples from 10 - months - old WT and MPS - IIIA mice injected with empty AAV2 / 9 vectors ( WT - empty and MPS - IIIA - empty experimental groups ) . Synaptosomal fractions were also blotted with anti - CSPα . ( C ) VAMP2 , SNAP - 25 and Syntaxin - 1 were immunoblotted in synaptosomal samples derived from the three experimental groups of mice . Protein levels were quantified . ( D ) The amount of SNARE - complexes was detected in synaptosomal brain samples derived from the three experimental groups of mice by immunoblotting analysis of non - boiled samples with VAMP2 , SNAP - 25 or Syntaxin - 1 antibodies . The amounts of SNARE complexes were quantified . ( E ) Total brain lysates derived from the three experimental groups of mice were immunoprecipitated with Syntaxin - 1 antibodies and co - immunoprecipitated VAMP2 and SNAP - 25 proteins were revealed by WB .

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Figure 7(A) Anti-myc immunostaining in different brain regions derived from 10-months-old MPS-IIIA mice intraventricularly injected with AAV2/9 vectors encoding CSPα under Synapsin-1 promoter (MPS-IIIA-myc-CSPα experimental group). As a control immunostaining was performed in brain sections from 10-months-old MPS-IIIA mice injected with empty AAV2/9 vectors (MPS-IIIA-empty experimental groups). To evaluate co-localization between exogenous CSPα and presynaptic compartment brain sections were also co-stained with anti-myc and anti-synapsin1 antibodies (confocal images are also shown).(B) Anti-myc WB in synaptosomal fractions derived from MPS-IIIA-myc-CSPα mice. As a control WB was performed in samples from 10-months-old WT and MPS-IIIA mice injected with empty AAV2/9 vectors (WT-empty and MPS-IIIA-empty experimental groups). Synaptosomal fractions were also blotted with anti-CSPα.(C) VAMP2, SNAP-25 and Syntaxin-1 were immunoblotted in synaptosomal samples derived from the three experimental groups of mice. Protein levels were quantified.(D) The amount of SNARE-complexes was detected in synaptosomal brain samples derived from the three experimental groups of mice by immunoblotting analysis of non-boiled samples with VAMP2, SNAP-25 or Syntaxin-1 antibodies. The amounts of SNARE complexes were quantified.(E) Total brain lysates derived from the three experimental groups of mice were immunoprecipitated with Syntaxin-1 antibodies and co-immunoprecipitated VAMP2 and SNAP-25 proteins were revealed by WB.


Figure 8 ( A ) EM analysis of cortical and hippocampal synapsis derived from the three experimental groups of mice . Synaptic vesicles number per synapse was quantified from ~ 40 different images ( taken from 5 mice for each group ) , normalized by the length of synaptic cleft and expressed as % of WT . The synaptic density was measured from 20 different images ( taken from 5 mice for each group ) as the number of synapses / area ( # / 500μm2 ) and expressed as % of WT . Arrows indicate the synaptic cleft while asterisks indicate abnormal vacuoles . Values are means ± s . e . m . * P < 0 . 05 , Student ' s t - test : Either MPS - IIIA - empty or MPS - IIIA - myc - CSPα vs WT - empty . Scale bar : 0 . 2 µm ( B ) Extracellular recordings ( fEPSPs ) in hippocampal brain slices from WT - empty ( n = 8 ) , MPS - IIIA - empty ( n = 5 ) and MPS - IIIA - myc - CSPα mice ( n = 5 ) . The upper panel shows representative fEPSP traces . Summary graph shows the fEPSPs slope as a function of the applied stimulus intensity . Data are means ± s . e . m . ; * P < 0 . 05 ( WT - empty vs MPS - IIIA - empty ) , # P < 0 . 05 ( MPS - IIIA - myc - CSPα vs . MPS - IIIA - empty ) , Kruskal - Wallis ANOVA followed by Dunn ' s test . ( C ) Mean distance travelled , maximal speed , immobility time and line crossing during 10 min testing in the open field , divided into 5 minute intervals in WT - empty , MPS - IIIA - empty and MPS - IIIA - myc - CSPα female mice ( n = 9 , n = 9 , n = 7 respectively ) . Distance , percentage open entries and open time in the elevated plus maze and latency to fall off the wire ( * body weight ) in the wire hanging test in WT - empty , MPS - IIIA - empty and MPS - IIIA - myc - CSPα male mice ( n = 10 , n = 8 , n = 9 respectively ) . Values are means ± s . e . m ; * P < 0 . 05 ( MPS - IIIA - empty vs . WT - empty ) , # P < 0 . 05 ( MPS - IIIA - myc - CSPα vs . MPS - IIIA - empty ) , two - way ANOVA for repeated measures for open field measures and t - test for the plus maze and the wire hanging followed by Duncan post - hoc test . Animals ' average age was 28 ± 2 weeks . ( D ) Neuronal cell death was evaluated in the cerebellum ( Calbindin immunostaining ) and in different layers of cortex ( NeuN immunostaining of frontal cortex ) of 10 months - old mice belonging to each experimental group of mice . Number of cells was quantified from 20 different images ( taken from 5 mice for each group ) and expressed as % of WT . Values are means ± s . e . m . * P < 0 . 05 , * * P < 0 . 001 Student ' s t - test . Scale bar : 50 µm . ( E ) Kaplan - Meier survival analysis in WT - empty ( n = 13 ) , MPS - IIIA - empty ( n = 13 ) and MPS - IIIA - myc - CSPα ( n = 13 ) male mice . The Kaplan - Meier survival curve was analyzed with the Chi - square test . A P value < 0 . 05 was considered to be statistically significant . P = 0 . 00003 ( MPS - IIIA - empty vs WT - empty ) , P = 0 . 000776 ( MPS - IIIA - myc - CSPα vs MPS - IIIA - empty ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) EM analysis of cortical and hippocampal synapsis derived from the three experimental groups of mice. Synaptic vesicles number per synapse was quantified from ~40 different images (taken from 5 mice for each group), normalized by the length of synaptic cleft and expressed as % of WT. The synaptic density was measured from 20 different images (taken from 5 mice for each group) as the number of synapses/area(#/500μm2) and expressed as % of WT. Arrows indicate the synaptic cleft while asterisks indicate abnormal vacuoles. Values are means ± s.e.m. *P<0.05, Student's t-test: Either MPS-IIIA-empty or MPS-IIIA-myc-CSPα vs WT-empty. Scale bar: 0.2 µm(B) Extracellular recordings (fEPSPs) in hippocampal brain slices from WT-empty (n=8), MPS-IIIA-empty (n=5) and MPS-IIIA-myc-CSPα mice (n=5). The upper panel shows representative fEPSP traces. Summary graph shows the fEPSPs slope as a function of the applied stimulus intensity. Data are means ± s.e.m.; *P < 0.05 (WT-empty vs MPS-IIIA-empty), #P < 0.05 (MPS-IIIA-myc-CSPα vs. MPS-IIIA-empty), Kruskal-Wallis ANOVA followed by Dunn's test.(C) Mean distance travelled, maximal speed, immobility time and line crossing during 10 min testing in the open field, divided into 5 minute intervals in WT-empty, MPS-IIIA-empty and MPS-IIIA-myc-CSPα female mice (n=9, n=9, n=7 respectively). Distance, percentage open entries and open time in the elevated plus maze and latency to fall off the wire (*body weight) in the wire hanging test in WT-empty, MPS-IIIA-empty and MPS-IIIA-myc-CSPα male mice (n=10, n=8, n=9 respectively). Values are means ± s.e.m; *P < 0.05 (MPS-IIIA-empty vs. WT-empty), #P < 0.05 (MPS-IIIA-myc-CSPα vs. MPS-IIIA-empty), two-way ANOVA for repeated measures for open field measures and t-test for the plus maze and the wire hanging followed by Duncan post-hoc test. Animals' average age was 28±2 weeks.(D) Neuronal cell death was evaluated in the cerebellum (Calbindin immunostaining) and in different layers of cortex (NeuN immunostaining of frontal cortex) of 10 months-old mice belonging to each experimental group of mice. Number of cells was quantified from 20 different images (taken from 5 mice for each group) and expressed as % of WT. Values are means ± s.e.m. *P<0.05, **P<0.001 Student's t-test. Scale bar: 50 µm.(E) Kaplan-Meier survival analysis in WT-empty (n=13), MPS-IIIA-empty (n=13) and MPS-IIIA-myc-CSPα (n=13) male mice. The Kaplan-Meier survival curve was analyzed with the Chi-square test. A P value <0.05 was considered to be statistically significant. P= 0.00003 (MPS-IIIA-empty vs WT-empty), P= 0.000776 (MPS-IIIA-myc-CSPα vs MPS-IIIA-empty).


Figure 1 . heparin . Reducing - end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface . PBS running buffer flow rate was 500 µL . min - 1 . The data for the three sensing channels are reported as an average response . Protein injections are delimited by the red arrows . A Injection of 100 nM FGF2 . heparin . Reducing - end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface . PBS running buffer flow rate was 500 µL . min - 1 . The data for the three sensing channels are reported as an average response . Protein injections are delimited by the red arrows . B 64 nM S1 RBD protein . C 64 nM S1 RBD protein over a streptavidin surface ( no heparin functionalization ) .

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Figure 1.heparin.Reducing-end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface. PBS running buffer flow rate was 500 µL.min-1. The data for the three sensing channels are reported as an average response. Protein injections are delimited by the red arrows. A Injection of 100 nM FGF2.heparin.Reducing-end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface. PBS running buffer flow rate was 500 µL.min-1. The data for the three sensing channels are reported as an average response. Protein injections are delimited by the red arrows. B 64 nM S1 RBD protein. C 64 nM S1 RBD protein over a streptavidin surface (no heparin functionalization).


Figure 2 . The structural change of the SARS - CoV - 2 S1 RBD observed in the presence of heparin by circular dichroism ( CD ) spectroscopy . ( A ) CD spectra of 200 μg / mL spike 1 alone ( purple ) or with 30 μg heparin ( orange ) in phosphate buffered saline pH 7 . 4 . Theoretical sum of spike 1 alone and 30 μg of heparin ( control ) if no interaction was observed ( black ) . ( B ) Δ secondary structure ( % ) of ( A ) . A 2 . 4 % increase in helix and 2 . 2 % decrease in antiparallel secondary structural features were calculated ( BestSel ) for the observed spectrum compared to that of the theoretical , summative spectrum of the SARS - CoV - 2 S1 RBD in the presence of heparin .

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Figure 2.The structural change of the SARS-CoV-2 S1 RBD observed in the presence of heparin by circular dichroism (CD) spectroscopy.(A) CD spectra of 200 μg/mL spike 1 alone (purple) or with 30 μg heparin (orange) in phosphate buffered saline pH 7.4. Theoretical sum of spike 1 alone and 30 μg of heparin (control) if no interaction was observed (black). (B) Δ secondary structure (%) of (A). A 2.4% increase in helix and 2.2% decrease in antiparallel secondary structural features were calculated (BestSel) for the observed spectrum compared to that of the theoretical, summative spectrum of the SARS-CoV-2 S1 RBD in the presence of heparin.


Figure 1 ( C , F ) Normalized average fluorescence profiles for mNG - PilG and mNG - PilH of hundreds to thousands of cells The length of each cell is normalized so that the dim pole is positioned at x = - 1 , the bright pole at x = 1 . The fluorescent profile for each cell is normalized to its total fluorescence . Solid lines : mean normalized fluorescence profiles across replicates . Shaded area , standard deviation across replicates .

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Figure 1(C,F) Normalized average fluorescence profiles for mNG-PilG and mNG-PilH of hundreds to thousands of cells The length of each cell is normalized so that the dim pole is positioned at x = -1, the bright pole at x = 1. The fluorescent profile for each cell is normalized to its total fluorescence. Solid lines: mean normalized fluorescence profiles across replicates. Shaded area, standard deviation across replicates.


Figure 2 ( A ) Fluorescence profiles of mNG - PilG in chpA mutants after 2h on surfaces . Solid lines , mean normalized fluorescence profiles across biological replicates . Shaded area , standard deviation across biological replicates . ( D ) Polar localization profiles of mNG - PilG in ΔfimL and ΔfimL ΔchpA mutant after 2h on surfaces .

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Figure 2(A) Fluorescence profiles of mNG-PilG in chpA mutants after 2h on surfaces. Solid lines, mean normalized fluorescence profiles across biological replicates. Shaded area, standard deviation across biological replicates.(D) Polar localization profiles of mNG-PilG in ΔfimL and ΔfimL ΔchpA mutant after 2h on surfaces.


Figure 3 ( A ) mNG - PilH fluorescence profiles in chpA mutants after 2h surface growth . To avoid negative effects of low cAMP level on localization of PilH , cpdA was deleted in all displayed strains to rescue cAMP level to WT levels Solid lines , mean normalized fluorescence profiles across biological replicates . Shaded area , standard deviation across biological replicates .

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Figure 3(A) mNG-PilH fluorescence profiles in chpA mutants after 2h surface growth. To avoid negative effects of low cAMP level on localization of PilH, cpdA was deleted in all displayed strains to rescue cAMP level to WT levels Solid lines, mean normalized fluorescence profiles across biological replicates. Shaded area, standard deviation across biological replicates.


Figure 4C ) Fluorescence profiles of mNG - PilHGOF after 2h on surface in chpA mutants . C ) Solid lines , mean normalized fluorescence profiles across biological replicates . Shaded area , standard deviation across biological replicates .

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Figure 4C) Fluorescence profiles of mNG-PilHGOF after 2h on surface in chpA mutants. C) Solid lines, mean normalized fluorescence profiles across biological replicates. Shaded area, standard deviation across biological replicates.


Figure 5Displacement maps derived from 50 randomly - selected cell trajectories from three random biological replicates . Each curve corresponds to an individual trajectory . A curve oriented to the top right corresponds to forward twitching , a curve oriented to the top left corresponds to reverse twitching . In each graph , we highlighted three representative tracks for clarity .

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Figure 5Displacement maps derived from 50 randomly-selected cell trajectories from three random biological replicates. Each curve corresponds to an individual trajectory. A curve oriented to the top right corresponds to forward twitching, a curve oriented to the top left corresponds to reverse twitching. In each graph, we highlighted three representative tracks for clarity.


Figure 6 ( A ) mNG - PilH fluorescence profiles in ΔpilG after 2h surface growth ( low cAMP ) . ( C ) mNG - PilH fluorescence profiles in ΔpilG ΔcpdA after 2h surface growth ( rescued cAMP ) .

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Figure 6(A) mNG-PilH fluorescence profiles in ΔpilG after 2h surface growth (low cAMP).(C) mNG-PilH fluorescence profiles in ΔpilG ΔcpdA after 2h surface growth (rescued cAMP).


Figure 7 ( A ) mNG - PilG fluorescence profiles in pilH mutants after 2h surface growth . We included ΔcpdA for comparison since both pilH mutations result in high cAMP levels . PilG is more polar and asymmetric in ΔpilH and pilHLOF . ( A Solid lines , mean normalized fluorescence profiles across biological replicates . Shaded area , standard deviation across biological replicates . ( D ) Time course of PilG localization in pilHGOF on surface . For clarity only the 2h profile of PilG in wild type was included . D ) Solid lines , mean normalized fluorescence profiles across biological replicates . Shaded area , standard deviation across biological replicates .

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Figure 7(A) mNG-PilG fluorescence profiles in pilH mutants after 2h surface growth. We included ΔcpdA for comparison since both pilH mutations result in high cAMP levels. PilG is more polar and asymmetric in ΔpilH and pilHLOF. (A Solid lines, mean normalized fluorescence profiles across biological replicates. Shaded area, standard deviation across biological replicates.(D) Time course of PilG localization in pilHGOF on surface. For clarity only the 2h profile of PilG in wild type was included. D) Solid lines, mean normalized fluorescence profiles across biological replicates. Shaded area, standard deviation across biological replicates.


Figure 8 ( A , Time course fluorescence profiles and polar localization indexes of mNG - PilG in ΔpilA . PilG polar localization index remains unchanged on surfaces in ΔpilA similar to surface - adapted WT ( C Time course fluorescence profiles and polar localization indexes of mNG - PilH in ΔpilA . Like in WT , PilH gets recruited to the poles over time in ΔpilA , although at a slower

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Figure 8(A, Time course fluorescence profiles and polar localization indexes of mNG-PilG in ΔpilA. PilG polar localization index remains unchanged on surfaces in ΔpilA similar to surface-adapted WT(C Time course fluorescence profiles and polar localization indexes of mNG-PilH in ΔpilA. Like in WT, PilH gets recruited to the poles over time in ΔpilA, although at a slower speed


Figure 1A . 3 - OH - C10 : 0 - induced ROS burst is suppressed by A23 and K252a . Two - week - old Col - 0 seedlings were treated with 2μM K252a or 40 μM A23 . One hour later , 1 μM 3 - OH - C10 : 0 was used to trigger the ROS burst , and the photo count was recorded by a luminometer . Mock is solvent control . RLU , relative light unit . Values are means ± SD ( n = 3 biological replicates ) . B . A23 and K252a suppress 3 - OH - C10 : 0 - induced PTI marker gene expression . Treatments with 3 - OH - C10 : 0 and inhibitors were same as in ( A ) , and the plant leaves were collected for RT - qPCR 4 hrs later . Values are means ± SD ( n = 3 biological replicates ) . ( ANOVA , P < 0 . 01 ) . C . MAPK cascade activation was inhibited by A23 . Treatments with 3 - OH - C10 : 0 and A23 were same as in ( A ) . The plant leaves were collected at 0 , 5 , and 10 min for immunoblotting . Activated MAPKs were detected by immunoblotting with anti - phospho - p44 / 42 MAPK antibody . The corresponding bands are indicated for MPK3 and MPK6 . Ponceau staining of Rubisco was used for estimating equal loading in each lane . The experiment was repeated three times with similar results . D . Tyrosine phosphorylation of LORE in vivo . Two - week - old 35S : LORE - FLAG complementation plants were subjected to immunoprecipitation with anti - FLAG agarose beads . A23 ( 40 μM ) was used to inhibit the protein phosphorylation , and the treatments were same as in ( A ) . Protein abundance of precipitated LORE - FLAG was detected by immunoblotting . The tyrosine phosphorylation was probed by anti - pTyrosine ( α - pY ) antibody . Similar results were observed in three biological replicates . E . LORE is an active tyrosine kinase . The α - pY antibody was used to probe tyrosine phosphorylation in recombinant protein MBP - tagged LORE - CD ( cytosolic domain ) . The LORE activation loop conserved K516 was mutated to E , and D613 was mutated to V . The bottom gel showing protein abundance stained by Coomassie Brilliant Blue ( CBB ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. 3-OH-C10:0-induced ROS burst is suppressed by A23 and K252a. Two-week-old Col-0 seedlings were treated with 2μM K252a or 40 μM A23. One hour later, 1 μM 3-OH-C10:0 was used to trigger the ROS burst, and the photo count was recorded by a luminometer. Mock is solvent control. RLU, relative light unit. Values are means ± SD (n=3 biological replicates).B. A23 and K252a suppress 3-OH-C10:0-induced PTI marker gene expression. Treatments with 3-OH-C10:0 and inhibitors were same as in (A), and the plant leaves were collected for RT-qPCR 4 hrs later. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01).C. MAPK cascade activation was inhibited by A23. Treatments with 3-OH-C10:0 and A23 were same as in (A). The plant leaves were collected at 0, 5, and 10 min for immunoblotting. Activated MAPKs were detected by immunoblotting with anti-phospho-p44/42 MAPK antibody. The corresponding bands are indicated for MPK3 and MPK6. Ponceau staining of Rubisco was used for estimating equal loading in each lane. The experiment was repeated three times with similar results.D. Tyrosine phosphorylation of LORE in vivo. Two-week-old 35S:LORE-FLAG complementation plants were subjected to immunoprecipitation with anti-FLAG agarose beads. A23 (40 μM) was used to inhibit the protein phosphorylation, and the treatments were same as in (A). Protein abundance of precipitated LORE-FLAG was detected by immunoblotting. The tyrosine phosphorylation was probed by anti-pTyrosine (α-pY) antibody. Similar results were observed in three biological replicates.E. LORE is an active tyrosine kinase. The α-pY antibody was used to probe tyrosine phosphorylation in recombinant protein MBP-tagged LORE-CD (cytosolic domain). The LORE activation loop conserved K516 was mutated to E, and D613 was mutated to V. The bottom gel showing protein abundance stained by Coomassie Brilliant Blue (CBB).