H : Urinary PAI - 1 measured by ELISA in a cohort of elderly patients with ( n = 48 ) or without ( n = 48 ) chronic kidney disease . Data information : Data are means ± SEM . Statistical analysis : t - student

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H: Urinary PAI-1 measured by ELISA in a cohort of elderly patients with (n = 48) or without (n = 48) chronic kidney disease. Data information: Data are means ± SEM. Statistical analysis: t-student test


Coomassie - stained SDS - PAGE gel showing purified protein from the predicted bacterial OTU constructs .

Coomassie-stained SDS-PAGE gel showing purified protein from the predicted bacterial OTU constructs.


Ub - PA activity - based probe assay for wild - type ( WT ) and catalytic Cys - to - Ala mutants ( CA ) . Strong , Cys - dependent reactivity is indicated with asterisks .

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Ub-PA activity-based probe assay for wild-type (WT) and catalytic Cys-to-Ala mutants (CA). Strong, Cys-dependent reactivity is indicated with asterisks.


Ub - KG ( TAMRA ) cleavage assay monitored by fluorescence polarization at the indicated DUB concentrations . Note that BurkOTU displays an increase in fluorescence polarization , indicative of noncovalent binding .

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Ub-KG(TAMRA) cleavage assay monitored by fluorescence polarization at the indicated DUB concentrations. Note that BurkOTU displays an increase in fluorescence polarization, indicative of noncovalent binding.


Heatmap representation of DUB activity against the Ub - KG ( TAMRA ) substrate shown in G . , including the WT enzyme and Ala substitutions at the predicted catalytic Cys , general base His , or acidic position . Substrate remaining at the end of the assay is reported after correction against an initial reading from an equivalent assay performed with the catalytically inactive CA mutants .

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Heatmap representation of DUB activity against the Ub-KG(TAMRA) substrate shown in G., including the WT enzyme and Ala substitutions at the predicted catalytic Cys, general base His, or acidic position. Substrate remaining at the end of the assay is reported after correction against an initial reading from an equivalent assay performed with the catalytically inactive CA mutants.


Ub / Ub - like specificity assay measuring activity of WT and inactive Cys - to - Ala wMelOTU toward the Ub - , ISG15 - , NEDD8 - , and SUMO1 - KG ( TAMRA ) substrates . Ub / Ub - like specificity assay measuring activity of WT and inactive Cys - to - Ala BurkOTU toward the Ub - , ISG15 - , NEDD8 - , and SUMO1 - KG ( TAMRA ) substrates . Note that the rise in fluorescence polarization signal , indicative of a noncovalent interaction , is specific to the Ub substrate .

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Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala wMelOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala BurkOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Note that the rise in fluorescence polarization signal, indicative of a noncovalent interaction, is specific to the Ub substrate.


Heatmap representation of corrected OTU activities toward the Ub and Ub - like fluorescent substrates . In the reactions marked by an asterisk , an unusually high level of noise in fluorescence polarization signal was observed , likely a result of high OTU concentration .

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Heatmap representation of corrected OTU activities toward the Ub and Ub-like fluorescent substrates. In the reactions marked by an asterisk, an unusually high level of noise in fluorescence polarization signal was observed, likely a result of high OTU concentration.


Ub chain specificity assay measuring wMelOTU activity toward the eight diUb linkages . Reaction samples were quenched at the indicated timepoints , resolved by SDS - PAGE , and visualized by Coomassie staining .

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Ub chain specificity assay measuring wMelOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS-PAGE, and visualized by Coomassie staining.


Ub chain specificity assay measuring BurkOTU activity toward the eight diUb linkages . Reaction samples were quenched at the indicated timepoints , resolved by SDS - PAGE , and visualized by Coomassie staining .

Ub chain specificity assay measuring BurkOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS-PAGE, and visualized by Coomassie staining.


Heatmap representation of WT bacterial OTU activities toward the eight diUb linkages at the indicated timepoints .

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Heatmap representation of WT bacterial OTU activities toward the eight diUb linkages at the indicated timepoints.


Ub - KG ( TAMRA ) cleavage assay monitoring the effects of structure - guided wMelOTU mutations . These data were collected in parallel with those presented in Fig . 1G and the WT dataset is shown again for reference .

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Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided wMelOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for reference.


Ub - KG ( TAMRA ) cleavage assay monitoring the effects of structure - guided EschOTU mutations . These data were collected in parallel with those presented in Fig . 1G and the WT dataset is shown again for clarity .

Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided EschOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for clarity.


Ub - PA activity - based probe assay for WT vOTU and sequence - permutated vOTUP . Strong reactivity is indicated with asterisks .

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Ub-PA activity-based probe assay for WT vOTU and sequence-permutated vOTUP. Strong reactivity is indicated with asterisks.


Ub - KG ( TAMRA ) cleavage assay monitored by fluorescence polarization for WT vOTU and sequence - permutated vOTUP .

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Ub-KG(TAMRA) cleavage assay monitored by fluorescence polarization for WT vOTU and sequence-permutated vOTUP.


About 25 μg of membrane ‐ enriched lysate from the mass spectrometry experiments was immunoblotted with anti ‐ PINK1 antibody . TOMM40 and GAPDH serve as markers for mitochondria and cytoplasm , respectively .

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About 25 μg of membrane‐enriched lysate from the mass spectrometry experiments was immunoblotted with anti‐PINK1 antibody. TOMM40 and GAPDH serve as markers for mitochondria and cytoplasm, respectively.


A - CConfirmation by mass spectrometry that Rab8A ( A ) , Rab8B ( B ) and Rab13 ( C ) Ser111 is phosphorylated upon PINK1 activation after CCCP treatment . Flp ‐ In T ‐ Rex HEK293 cells expressing empty ‐ FLAG , WT PINK1 ‐ FLAG and KI ( D384A ) PINK1 ‐ FLAG were transfected either with HA ‐ Rab8A ( A ) , HA ‐ Rab8B ( B ) or HA ‐ Rab13 ( C ) induced with doxycycline and stimulated with 10 μM of CCCP for 3 h . Whole ‐ cell lysates ( 10 mg ) were immunoprecipitated with anti ‐ HA agarose , resolved by SDS - PAGE and stained with colloidal Coomassie blue ( second panel ) . Coomassie ‐ stained bands migrating with expected molecular mass of HA ‐ Rabs were excised , in ‐ gel digested with trypsin and subjected to high ‐ performance liquid chromatography with LC ‐ MS / MS on an LTQ ‐ Orbitrap mass spectrometer . Upper panel shows the extracted ion chromatogram ( XIC ) analysis of Ser111 ‐ containing phosphopeptides ( 8A , NIEEHApSADVEK ; 8B , NIEEHApSSDVER ; 13 , SIKENApSAGVER ) with the combined signal intensity of the 2 + and 3 + forms of the peptide indicated on the y ‐ axis . Note that the Ser111 phosphopeptide was only detected in samples from WT PINK1 ‐ FLAG ‐ expressing cells following CCCP stimulation .

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A-CConfirmation by mass spectrometry that Rab8A (A), Rab8B (B) and Rab13 (C) Ser111 is phosphorylated upon PINK1 activation after CCCP treatment. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected either with HA‐Rab8A (A), HA‐Rab8B (B) or HA‐Rab13 (C) induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (10 mg) were immunoprecipitated with anti‐HA agarose, resolved by SDS-PAGE and stained with colloidal Coomassie blue (second panel). Coomassie‐stained bands migrating with expected molecular mass of HA‐Rabs were excised, in‐gel digested with trypsin and subjected to high‐performance liquid chromatography with LC‐MS/MS on an LTQ‐Orbitrap mass spectrometer. Upper panel shows the extracted ion chromatogram (XIC) analysis of Ser111‐containing phosphopeptides (8A, NIEEHApSADVEK; 8B, NIEEHApSSDVER; 13, SIKENApSAGVER) with the combined signal intensity of the 2+ and 3+ forms of the peptide indicated on the y‐axis. Note that the Ser111 phosphopeptide was only detected in samples from WT PINK1‐FLAG‐expressing cells following CCCP stimulation.


DCharacterisation of Rab8A , Rab8B and Rab13 phospho ‐ Ser111 antibodies . Flp ‐ In T ‐ Rex HEK293 cells expressing empty ‐ FLAG , WT PINK1 ‐ FLAG and KI ( D384A ) PINK1 ‐ FLAG were transfected with either WT or Ser111Ala ‐ mutant ( S111A ) HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 , induced with doxycycline and stimulated with 10 μM of CCCP for 3 h . Whole ‐ cell lysates ( 0 . 25 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with Rab8A , Rab8B or Rab13 phospho ‐ Ser111 antibodies . Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls .

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DCharacterisation of Rab8A, Rab8B and Rab13 phospho‐Ser111 antibodies. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected with either WT or Ser111Ala‐mutant (S111A) HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls.


PINK1 is essential for CCCP ‐ mediated Rab8A Ser111 phosphorylation . WT and PINK1 KO HeLa cells were transfected with either WT or Ser111Ala ( S111A ) mutant constructs of HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 . Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1 ‐ 3xFLAG or KI ( D384A ) PINK1 ‐ 3xFLAG as indicated . After transfection for at least 24 h , cells were treated with DMSO as a vehicle control or CCCP for 20 h . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with Rab8A , Rab8B or Rab13 phospho ‐ Ser111 antibody . Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls . For the lower panel , whole ‐ cell lysates ( 30 μg ) were immunoblotted with total PINK1 antibody to confirm PINK1 expression and with GAPDH as loading controls .

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PINK1 is essential for CCCP‐mediated Rab8A Ser111 phosphorylation. WT and PINK1 KO HeLa cells were transfected with either WT or Ser111Ala (S111A) mutant constructs of HA‐Rab8A, HA‐Rab8B or HA‐Rab13. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated. After transfection for at least 24 h, cells were treated with DMSO as a vehicle control or CCCP for 20 h. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with total PINK1 antibody to confirm PINK1 expression and with GAPDH as loading controls.


Endogenous Rab8A Ser111 phosphorylation is PINK1 dependent . WT and PINK1 KO HeLa cells were treated with DMSO as a vehicle control or CCCP for 20 h . Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1 ‐ 3xFLAG or KI ( D384A ) PINK1 ‐ 3xFLAG as indicated for at least 24 h before CCCP treatment . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ Rab8A pre ‐ bound with protein A agarose followed by immunoblot with Rab8A phospho ‐ Ser111 antibody . Part of the immunoprecipitates was used to immunoblot with anti ‐ total Rab8A antibody as loading controls .

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Endogenous Rab8A Ser111 phosphorylation is PINK1 dependent. WT and PINK1 KO HeLa cells were treated with DMSO as a vehicle control or CCCP for 20 h. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated for at least 24 h before CCCP treatment. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A pre‐bound with protein A agarose followed by immunoblot with Rab8A phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot with anti‐total Rab8A antibody as loading controls.


Absence of Rab8A Ser111 phosphorylation in human mutant PINK1 patient fibroblasts . Primary skinfibroblasts were derived from a patient with homozygous PINK1 Q456X mutation or unaffected control . Cells were incubated with DMSO or CCCP for 20 h , and whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho ‐ Ser111 antibody . Lysates ( 1 mg ) were also subjected to immunoprecipitation with polyclonal anti ‐ PINK1 antibody and immunoblotted with monoclonal PINK1 antibody . Equal loading of protein extracts was confirmed by GAPDH .

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Absence of Rab8A Ser111 phosphorylation in human mutant PINK1 patient fibroblasts. Primary skinfibroblasts were derived from a patient with homozygous PINK1 Q456X mutation or unaffected control. Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with polyclonal anti‐PINK1 antibody and immunoblotted with monoclonal PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.


Absence of Rab8A Ser111 phosphorylation in PINK1 knockout MEFs . MEFs were derived from PINK1 knockout embryos or wild ‐ type controls ( see Materials and Methods ) . Cells were incubated with DMSO or CCCP for 20 h , and whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho ‐ Ser111 antibody . Lysates ( 1 mg ) were also subjected to immunoprecipitation with a polyclonal anti ‐ mouse ‐ specific PINK1 antibody and immunoblotted with a different anti ‐ mouse ‐ specific PINK1 antibody . Equal loading of protein extracts was confirmed by GAPDH .

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Absence of Rab8A Ser111 phosphorylation in PINK1 knockout MEFs. MEFs were derived from PINK1 knockout embryos or wild‐type controls (see Materials and Methods). Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with a polyclonal anti‐mouse‐specific PINK1 antibody and immunoblotted with a different anti‐mouse‐specific PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.


Rab8A is not required for the activation of Parkin E3 ligase activity at mitochondria in response to PINK1 activation by CCCPWild ‐ type ( WT ) or Rab8A knockout ( KO ) HeLa cells were transfected with WT or Cys431Phe ( C341F ) ‐ mutant Parkin . After transfection for 24 h , cells were treated with DMSO as a vehicle control or 10 μM CCCP for 6 h . Mitochondrial enriched extracts ( mitochondrial lysate ) were incubated with ubiquitin ‐ binding resins derived from his ‐ halo ‐ ubiquilin1 UBA domain tetramer ( UBAUBQLN1 ) . Captured ubiquitylated proteins were subject to immunoblotting with anti ‐ CISD1 and anti ‐ mitofusin 2 antibodies . Mitochondrial lysate and total lysate were also subjected to immunoblotting with indicated antibodies for loading and protein expression controls . Phospho ‐ Ser111 Rab8A was detected after Rab8A immunoprecipitation from 200 μg of mitochondrial lysate with anti ‐ Rab8A antibody .

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Rab8A is not required for the activation of Parkin E3 ligase activity at mitochondria in response to PINK1 activation by CCCPWild‐type (WT) or Rab8A knockout (KO) HeLa cells were transfected with WT or Cys431Phe (C341F)‐mutant Parkin. After transfection for 24 h, cells were treated with DMSO as a vehicle control or 10 μM CCCP for 6 h. Mitochondrial enriched extracts (mitochondrial lysate) were incubated with ubiquitin‐binding resins derived from his‐halo‐ubiquilin1 UBA domain tetramer (UBAUBQLN1). Captured ubiquitylated proteins were subject to immunoblotting with anti‐CISD1 and anti‐mitofusin 2 antibodies. Mitochondrial lysate and total lysate were also subjected to immunoblotting with indicated antibodies for loading and protein expression controls. Phospho‐Ser111 Rab8A was detected after Rab8A immunoprecipitation from 200 μg of mitochondrial lysate with anti‐Rab8A antibody.


In vitro phosphorylation analysis of Rab8A by PINK1 . WT or S111A ‐ mutant Rab8A ( 1 . 2 μg ) was incubated in the presence of MBP ‐ fused WT or KI ( D359A ) TcPINK1 ( 1 . 1 μg ) and Mg2 + ‐ [ γ ‐ 32P ] ATP for the indicated time . Samples were subjected to SDS - PAGE , and proteins were detected by colloidal Coomassie blue staining ( lower panel ) . The [ γ ‐ 32P ] incorporation to substrate was detected by autoradiography ( upper panel ) . Cerenkov counting was used to calculate the stoichiometry of substrate phosphorylation as mol of [ γ ‐ 32P ] incorporation / mol of substrate . Ubiquitin was used as a positive control of the TcPINK1 substrate .

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In vitro phosphorylation analysis of Rab8A by PINK1. WT or S111A‐mutant Rab8A (1.2 μg) was incubated in the presence of MBP‐fused WT or KI (D359A) TcPINK1 (1.1 μg) and Mg2+‐[γ‐32P] ATP for the indicated time. Samples were subjected to SDS-PAGE, and proteins were detected by colloidal Coomassie blue staining (lower panel). The [γ‐32P] incorporation to substrate was detected by autoradiography (upper panel). Cerenkov counting was used to calculate the stoichiometry of substrate phosphorylation as mol of [γ‐32P] incorporation/mol of substrate. Ubiquitin was used as a positive control of the TcPINK1 substrate.


Time ‐ course comparison of PINK1 ‐ mediated Rab8A , Rab8B and Rab13 Ser111 phosphorylation vs . Parkin Ser65 phosphorylation . Flp ‐ In T ‐ Rex HEK293 cells expressing WT PINK1 ‐ FLAG were transfected with either WT or Ser111Ala ‐ ( S111A ) mutant HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 , induced with doxycycline and stimulated with CCCP for the indicated time . In parallel , Flp ‐ In T ‐ Rex HEK293 cells expressing WT PINK1 ‐ FLAG were transfected with either WT or Ser65 Ala ( S65A ) ‐ mutant Parkin . Whole ‐ cell lysates ( 0 . 25 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with indicated phospho ‐ Ser111 antibodies . Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls . For the lower panel , whole ‐ cell lysates ( 30 μg ) were immunoblotted with indicated antibodies .

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Time‐course comparison of PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser111Ala‐(S111A) mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with CCCP for the indicated time. In parallel, Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser65 Ala (S65A)‐mutant Parkin. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.


Time ‐ course comparison of endogenous PINK1 ‐ mediated Rab8A , Rab8B and Rab13 Ser111 phosphorylation vs . Parkin Ser65 phosphorylation . HeLa cells were transfected with either WT or S111A ‐ mutant HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 for at least 24 h before CCCP treatment for the indicated time . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with indicated phospho ‐ Ser111 antibodies . In parallel , HeLa cells were transfected with either WT or S65A ‐ mutant Parkin and whole ‐ cell lysates ( 30 μg ) were immunoblotted with indicated antibodies .

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Time‐course comparison of endogenous PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. HeLa cells were transfected with either WT or S111A‐mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13 for at least 24 h before CCCP treatment for the indicated time. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. In parallel, HeLa cells were transfected with either WT or S65A‐mutant Parkin and whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.


Rabin8 ‐ catalysed mant ‐ GDP release from mant ‐ GDP loaded WT , S111A and S111E mutants of Rab8A . The Rab proteins ( 1 μM ) were incubated with 100 μM GDP in buffer ( 20 mM HEPES pH 7 . 5 , 50 mM NaCl , 1 mM MgCl2 , 2 mM DTE ) , and the reaction was started by the addition of 0 . 5 μM Rabin8 . The decrease in mant fluorescence was used as a measure of mantGDP release .

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Rabin8‐catalysed mant‐GDP release from mant‐GDP loaded WT, S111A and S111E mutants of Rab8A. The Rab proteins (1 μM) were incubated with 100 μM GDP in buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 1 mM MgCl2, 2 mM DTE), and the reaction was started by the addition of 0.5 μM Rabin8. The decrease in mant fluorescence was used as a measure of mantGDP release.


Rab8A Ser111 phosphorylation impairs Rabin8 interaction in cells . Rab8A KO HeLa cells were transfected with wild ‐ type ( WT ) , S111E or S111A HA ‐ Rab8A . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with Rabin8 or anti ‐ HA antibody . Lysates were immunoblotted with Rabin8 or anti ‐ HA antibody to confirm equivalent expression of Rabin8 and WT and mutant HA ‐ Rab8A in extracts .

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Rab8A Ser111 phosphorylation impairs Rabin8 interaction in cells. Rab8A KO HeLa cells were transfected with wild‐type (WT), S111E or S111A HA‐Rab8A. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rabin8 or anti‐HA antibody. Lysates were immunoblotted with Rabin8 or anti‐HA antibody to confirm equivalent expression of Rabin8 and WT and mutant HA‐Rab8A in extracts.


B Mass spectrometry analysis of proteins co - purified with Ccq1HA . Shown is a volcano plot for proteins significantly enriched in Ccq1HA relative to untagged Ccq1 ( left panel : WT cells ; right panel : rik1 ∆ cells ) . Members of the CLRC complex and shelterin are highlighted in red and blue , respectively . Bottom panel displays proteins enriched ( log2 ≥ 5 or pval ≤ 0 . 01 )

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B Mass spectrometry analysis of proteins co-purified with Ccq1HA. Shown is a volcano plot for proteins significantly enriched in Ccq1HA relative to untagged Ccq1 (left panel: WT cells; right panel: rik1∆ cells). Members of the CLRC complex and shelterin are highlighted in red and blue, respectively. Bottom panel displays proteins enriched (log2 ≥ 5 or pval ≤ 0.01)


ChIP - qPCR analysis of epitope - tagged shelterin components in WT and mutant strains as indicated ( negative control : untagged strain ) . Positions on x - axis denote distances relative to telomeric repeats ( telomere - proximal PCR primer ) ( n = 3 independent experiments ) Data information data are normalized to input and represented as mean ±

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ChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEM


F ChIP analysis of Ccq1HA and FLAG - TetR - Clr4 * ( see scheme ) . Expression and tethering of FLAG - TetR - Clr4 * are controlled by thiamine / AHT addition ( red dots indicate induced / tethered FLAG - TetR - Clr4 * ; empty circles indicate non - induced / non - bound controls ) ( n = 3 independent experiments ) Data information data are normalized to input and represented as mean ±

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F ChIP analysis of Ccq1HA and FLAG-TetR-Clr4* (see scheme). Expression and tethering of FLAG-TetR-Clr4* are controlled by thiamine/AHT addition (red dots indicate induced/tethered FLAG-TetR-Clr4*; empty circles indicate non-induced/non-bound controls) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEM


A Left : Schemes of CLRC and the shelterin complex . Middle and right : Scheme depicting the position of the ura4 + reporter gene and heterochromatin islands relative to telomeric repeats and TAS regions ; note that the minichromosomesome Ch16 does not contain TAS . B - C ChIP - qPCR analyses of H3K9me2 ( B ) and H3 ( C ) in WT and ccq1Δ cells ( n = 3 independent experiments . D Fold change of WT over ccq1Δ for H3K9me2 and H3 level Data information : In ( B , C ) , data are normalized to input and to the average of three euchromatic loci ( EC ) as internal contro and represented as mean ±

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A Left: Schemes of CLRC and the shelterin complex. Middle and right: Scheme depicting the position of the ura4+ reporter gene and heterochromatin islands relative to telomeric repeats and TAS regions; note that the minichromosomesome Ch16 does not contain TAS. B-C ChIP-qPCR analyses of H3K9me2 (B) and H3 (C) in WT and ccq1Δ cells (n = 3 independent experiments. D Fold change of WT over ccq1Δ for H3K9me2 and H3 level Data information: In (B, C), data are normalized to input and to the average of three euchromatic loci (EC) as internal contro and represented as mean ± SEM


ChIP - qPCR analyses of H3 in WT and mutants as indicated ( n = 3 independent experiments ) . Data information data are represented as mean ±

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ChIP-qPCR analyses of H3 in WT and mutants as indicated (n = 3 independent experiments). Data information data are represented as mean ± SE


ChIP - qPCR analyses of H3K9me2 in WT and mutants as indicated ( n = 3 independent experiments ) . Data information data are represented as mean ±

ChIP-qPCR analyses of H3K9me2 in WT and mutants as indicated (n = 3 independent experiments). Data information data are represented as mean ± SE


A ChIP - seq reads mapped to TEL2R ( shown in reverse orientation for consistency )

A ChIP-seq reads mapped to TEL2R (shown in reverse orientation for consistency)


A ChIP - qPCR analysis of H3 at endogenous TAS and a fragment spanning the TAS region from 115 - 905 bp ( relative to telomeric repeat ) inserted into the leu1 + locus ( see scheme ) . The TAS fragment was inserted into a strain that lacks endogenous TA Shown are ChIP analyses for WT ( left ) and ccq1 ∆ ( right ; note different the scale of the y - axis ) ( n = 9 - 10 independent experiments except for ectopic TAS in ccq1 ∆ strain where n = 3 ) Data information data are represented as mean ±

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A ChIP-qPCR analysis of H3 at endogenous TAS and a fragment spanning the TAS region from 115-905 bp (relative to telomeric repeat) inserted into the leu1+ locus (see scheme). The TAS fragment was inserted into a strain that lacks endogenous TA Shown are ChIP analyses for WT (left) and ccq1∆ (right; note different the scale of the y-axis) (n = 9-10 independent experiments except for ectopic TAS in ccq1∆ strain where n = 3) Data information data are represented as mean ± SE


B ChIP - qPCR analysis of H3 at reporter genes ( ura4 + and his3 + ) at various chromosomal locations ( see schemes ) in WT cells . TAS1 , TAS2 , and TAS3 correspond to position 116 bp , 2851 bp and 6291 bp ( relative to telomeric repeats ) , respectively ( n = 3 independent experiments ) . Data information data are represented as mean ±

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B ChIP-qPCR analysis of H3 at reporter genes (ura4+ and his3+) at various chromosomal locations (see schemes) in WT cells. TAS1, TAS2, and TAS3 correspond to position 116 bp, 2851 bp and 6291 bp (relative to telomeric repeats), respectively (n = 3 independent experiments). Data information data are represented as mean ± SE


A , B RT - qPCR analysis of transcript levels of TERRA ( A ) and tlh1 + ( B ) in WT strain and mutants as indicated ( double mutants with ccq1Δ are indicated by blue dot ) . Data information : In ( A , B ) , data is represented as mean ± SEM from 3 independent experiments and shown relative to WT

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A, B RT-qPCR analysis of transcript levels of TERRA (A) and tlh1+ (B) in WT strain and mutants as indicated (double mutants with ccq1Δ are indicated by blue dot). Data information: In (A, B), data is represented as mean ± SEM from 3 independent experiments and shown relative to WT leve


A Genomic copy number of his3 + , ura4 + and TAS1 in indicated strains harboring the reporter genes tel1L : : his3 + and tel2L : : ura4 + . Cultures from individual WT and freshly generated knockout clones ( ccq1Δ , n = 16 ; clr4Δ , n = 5 ; ccq1Δ clr4Δ , n = 5 ) were pre - grown on selective media for several days and inoculated at day 0 to grow in liquid media with regular back - dilution ( every 24 hours , approximately 7 generations ) . Samples were taken at indicated harvest times , and relative copy numbers of genomic regions were assessed by qPCR ( normalization against intrachromosomal loci ) . Black and rainbow color lines indicate WT strains and individual ccq1 + deletion mutants ( clones ) ,

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A Genomic copy number of his3+, ura4+ and TAS1 in indicated strains harboring the reporter genes tel1L::his3+ and tel2L::ura4+. Cultures from individual WT and freshly generated knockout clones (ccq1Δ, n = 16; clr4Δ, n = 5; ccq1Δ clr4Δ, n = 5) were pre-grown on selective media for several days and inoculated at day 0 to grow in liquid media with regular back-dilution (every 24 hours, approximately 7 generations). Samples were taken at indicated harvest times, and relative copy numbers of genomic regions were assessed by qPCR (normalization against intrachromosomal loci). Black and rainbow color lines indicate WT strains and individual ccq1+ deletion mutants (clones), respectively


B qPCR analysis as in ( A ) but with strains harboring the minichromosome Ch16 m23 : : ura4 + ( ccq1Δ , n = 14 ) . Since Ch16 is unstable in ccq1Δ cell Note that Ch16 does not harbor TAS

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B qPCR analysis as in (A) but with strains harboring the minichromosome Ch16 m23::ura4+ (ccq1Δ, n = 14). Since Ch16 is unstable in ccq1Δ cell Note that Ch16 does not harbor TAS sequences


Representative HDX - MS curves showing the protected regions in CD53 and CD2 . The HDX experiments were performed ( manually or with a LEAP automation system ) in triplicate . Error bars show the standard deviation .

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Representative HDX-MS curves showing the protected regions in CD53 and CD2. The HDX experiments were performed (manually or with a LEAP automation system) in triplicate. Error bars show the standard deviation.


D Representative HDX - MS curves showing the protected regions in CD81 and CD19 . The HDX experiments were manually performed in triplicate . Error bars show the standard deviation .

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D Representative HDX-MS curves showing the protected regions in CD81 and CD19. The HDX experiments were manually performed in triplicate. Error bars show the standard deviation.


A CD81 knockout of 293T cells . The knockout cell line was generated by CRISPR - Cas9 . The immunoblots using CD81 antibody and actin antibody were conducted on the same film .

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A CD81 knockout of 293T cells. The knockout cell line was generated by CRISPR-Cas9. The immunoblots using CD81 antibody and actin antibody were conducted on the same film.


B CD81 facilitates CD19 maturation . CD81 and CD19 ( 300 ng DNA ) were transfected into the CD81 - knockout 293T cells . Anti - FLAG immunoblot was performed for CD19 with a C - FLAG tag . The deglycosylation was performed by digesting the cell lysate with PNGase F or Endo H , following the manufacturers ' instructions . The deglycosylation pattern of CD19 species ( p : precursor , m and m ' : two mature forms ) differs because the glycosylation content of these species is

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B CD81 facilitates CD19 maturation. CD81 and CD19 (300 ng DNA) were transfected into the CD81-knockout 293T cells. Anti-FLAG immunoblot was performed for CD19 with a C-FLAG tag. The deglycosylation was performed by digesting the cell lysate with PNGase F or Endo H, following the manufacturers' instructions. The deglycosylation pattern of CD19 species (p: precursor, m and m': two mature forms) differs because the glycosylation content of these species is different


C Trypsin digestion of CD19 . The digestion was performed at 37 ° C with intact cells . The mature CD19 forms ( m and m ' ) can be digested because they are surface expressed , whereas the intracellular precursor form ( p ) is protected from the digestion .

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C Trypsin digestion of CD19. The digestion was performed at 37 °C with intact cells. The mature CD19 forms (m and m') can be digested because they are surface expressed, whereas the intracellular precursor form (p) is protected from the digestion.


E EC1 mutants of CD81 interfere with the accurate glycosylation of CD19 . Chol : mutations at the cholesterol - binding site of CD81 . CD81 constructs and CD19 ( 300 ng DNA ) were co - transfected into the CD81 - knockout 293T cells . Top , Anti - FLAG immunoblot was performed for CD19 with a C - FLAG tag . In presence of wildtype CD81 ( wt ) , most CD19 is in the properly glycosylated mature form ( m ) , and the overglycosylated m ' form is not observed . With the EC1 mutants ( A4 in particular ) , however , m ' emerges and m shifts to high MW , indicating an alteration in the CD19 glycosylation . The level of intracellular p form is low with the wildtype CD81 or with the mutants . Bottom , Control experiment showing similar expression level of wild - type CD81 and CD81 mutants . Anti - V5 immunoblotting was conducted for the CD81 constructs with a C - V5 tag , and anti - actin immunoblotting was conducted on the same film .

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E EC1 mutants of CD81 interfere with the accurate glycosylation of CD19. Chol: mutations at the cholesterol-binding site of CD81. CD81 constructs and CD19 (300 ng DNA) were co-transfected into the CD81-knockout 293T cells. Top, Anti-FLAG immunoblot was performed for CD19 with a C-FLAG tag. In presence of wildtype CD81 (wt), most CD19 is in the properly glycosylated mature form (m), and the overglycosylated m' form is not observed. With the EC1 mutants (A4 in particular), however, m' emerges and m shifts to high MW, indicating an alteration in the CD19 glycosylation. The level of intracellular p form is low with the wildtype CD81 or with the mutants. Bottom, Control experiment showing similar expression level of wild-type CD81 and CD81 mutants. Anti-V5 immunoblotting was conducted for the CD81 constructs with a C-V5 tag, and anti-actin immunoblotting was conducted on the same film.


A Migration of naïve Cd53 - / - cultured primary pre - B cells to 100 ng / mL CXCL12 compared to WT littermate controls ( n = 5 mice per group ) over three independent experiments . Bars and error bars represent mean ± standard error of the mean . * * p < 0 . 01 by two - way ANOVA .

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A Migration of naïve Cd53-/- cultured primary pre-B cells to 100 ng/mL CXCL12 compared to WT littermate controls (n = 5 mice per group) over three independent experiments. Bars and error bars represent mean ± standard error of the mean. **p < 0.01 by two-way ANOVA.


B Migration of Cd53 - / - pre - B cells transduced with empty , control , or mutant constructs in response to CXCL12 gradient , 3 days after transduction ( n = 2 - 5 mice per group ) over two independent experiments . Bars and error bars represent mean ± standard error of the mean . * * p < 0 . 01 and * * * p < 0 . 001 by two - way ANOVA .

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B Migration of Cd53-/- pre-B cells transduced with empty, control, or mutant constructs in response to CXCL12 gradient, 3 days after transduction (n = 2-5 mice per group) over two independent experiments. Bars and error bars represent mean ± standard error of the mean. **p < 0.01 and ***p < 0.001 by two-way ANOVA.


A . LN229 and U87 cells treated with Wnt 3a ( 0 , 10 , 20 , 50 , 100 ng / ml ) for 4 hr . The indicated proteins were analyzed by Western blotting .

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A. LN229 and U87 cells treated with Wnt 3a (0, 10, 20, 50, 100 ng/ml) for 4 hr. The indicated proteins were analyzed by Western blotting.


B . LN229 cells treated with or without Wnt3a ( 50 ng / ml ) were pulsed with [ 35S ] methionine for 30 min and chased for the times indicated . Protein extracts were used for IP with FoxM1 antibodies and subjected to SDS - PAGE and autoradiography . The intensities of the FoxM1 bands in autoradiography were then quantified by using NIH Image software , and the densities of the FoxM1 bands at time 0 were set as 100 % . Values are mean ± SD from two independent experiments .

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B. LN229 cells treated with or without Wnt3a (50 ng/ml) were pulsed with [35S] methionine for 30 min and chased for the times indicated. Protein extracts were used for IP with FoxM1 antibodies and subjected to SDS-PAGE and autoradiography. The intensities of the FoxM1 bands in autoradiography were then quantified by using NIH Image software, and the densities of the FoxM1 bands at time 0 were set as 100%. Values are mean ± SD from two independent experiments.


C . Levels of FoxM1 were analyzed by Western blotting in LN229 and U87 cells treated with Wnt inhibitor DKK1 ( 100 ng / ml ) for indicated times . Noted that the basal levels of FoxM1 and β - catenin in ( A ) and ( C ) are similar but look different due to variations on the exposure time of the Western blots .

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C. Levels of FoxM1 were analyzed by Western blotting in LN229 and U87 cells treated with Wnt inhibitor DKK1 (100 ng/ml) for indicated times. Noted that the basal levels of FoxM1 and β-catenin in (A) and (C) are similar but look different due to variations on the exposure time of the Western blots.


D . FoxM1 expression levels were determined in LN229 cells treated with or without GSK3 inhibitor LiCl ( 10 mM ) and CHX ( 100 µg / ml ) for indicated times .

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D. FoxM1 expression levels were determined in LN229 cells treated with or without GSK3 inhibitor LiCl (10 mM) and CHX (100 µg/ml) for indicated times.


E . GSK3 wild - type ( WT ) and knockout ( KO ) cells were treated with CHX ( 100 µg / ml ) for indicated times , and FoxM1 and GSK3α / β expression levels were determined by Western blotting .

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E. GSK3 wild-type (WT) and knockout (KO) cells were treated with CHX (100 µg/ml) for indicated times, and FoxM1 and GSK3α/β expression levels were determined by Western blotting.


F . Flag - FoxM1 and GSK3βKA or GSK3βKD plasmids were co - transfected into 293T cells for 48 hr . The cells were then treated with CHX ( 100 µg / ml ) for indicated times . Exogenous Flag - FoxM1 expression level was determined by Western blotting .

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F. Flag-FoxM1 and GSK3βKA or GSK3βKD plasmids were co-transfected into 293T cells for 48 hr. The cells were then treated with CHX (100 µg/ml) for indicated times. Exogenous Flag-FoxM1 expression level was determined by Western blotting.


B . Lysates from 293T cells expressing Flag - FoxM1 or its mutants were subjected to IP using mouse anti - Flag antibody , followed by IB with GSK3α / β antibody ( upper panel ) and rabbit anti - Flag antibody ( middle panel ) .

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B. Lysates from 293T cells expressing Flag-FoxM1 or its mutants were subjected to IP using mouse anti-Flag antibody, followed by IB with GSK3α/β antibody (upper panel) and rabbit anti-Flag antibody (middle panel).


D . HA - ubiquitin and Flag - tagged FoxM1 ( WT ) or mutants FoxM1S228A , FoxM1T309A , or Flag - FoxM1S474A plasmid were co - transfected into 293T cells . After 36 hr , cells were treated with 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with Flag antibody , followed by IB with Flag and HA antibody .

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D. HA-ubiquitin and Flag-tagged FoxM1 (WT) or mutants FoxM1S228A, FoxM1T309A, or Flag-FoxM1S474A plasmid were co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag antibody, followed by IB with Flag and HA antibody.


E . HA - ubiquitin was co - transfected into 293T cells . After 36 hr , cells were treated with 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with IgG or FoxM1 phospho - S474 antibody , followed by IB with FoxM1 and HA antibody .

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E. HA-ubiquitin was co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 phospho-S474 antibody, followed by IB with FoxM1 and HA antibody.


F . HA - tagged K48 - only ubiquitin or K63 - only ubiquitin construct was transfected into 293T cells . After 36 hr , cells were treated with 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with IgG or FoxM1 antibody , followed by IB with FoxM1 and HA antibody .

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F. HA-tagged K48-only ubiquitin or K63-only ubiquitin construct was transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 antibody, followed by IB with FoxM1 and HA antibody.


G . GSK3βCA or vector and Flag - tagged FoxM1 ( WT ) , mutants S474A or S478A were co - transfected into 293T cells . Lysates of the cells were subjected to IP with Flag antibody and followed by IB with FoxM1 phospho - S474 antibody .

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G. GSK3βCA or vector and Flag-tagged FoxM1 (WT), mutants S474A or S478A were co-transfected into 293T cells. Lysates of the cells were subjected to IP with Flag antibody and followed by IB with FoxM1 phospho-S474 antibody.


A . Cell lysates of LN229 cells were subjected to IP using Axin antibody or control IgG , followed by IB with indicated antibodies .

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A. Cell lysates of LN229 cells were subjected to IP using Axin antibody or control IgG, followed by IB with indicated antibodies.


B . Axin siRNA or control siRNA were transfected into LN229 cells , after 36 hours , lysate of the cells was performed with co - IP using FoxM1 antibody followed by IB with indicated antibodies .

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B. Axin siRNA or control siRNA were transfected into LN229 cells, after 36 hours, lysate of the cells was performed with co-IP using FoxM1 antibody followed by IB with indicated antibodies.


C . Axin siRNA or control siRNA were transfected into LN229 cells for 36 hours . Lysates of the cells were subjected to IB with FoxM1 phospho - S474 antibody .

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C. Axin siRNA or control siRNA were transfected into LN229 cells for 36 hours. Lysates of the cells were subjected to IB with FoxM1 phospho-S474 antibody.


D . Cell lysates of LN229 cells with or without Wnt - 3a treatment for 4 hours were subjected to IP using Axin antibody or control IgG , followed by IB with FoxM1 and Axin antibodies . .

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D. Cell lysates of LN229 cells with or without Wnt-3a treatment for 4 hours were subjected to IP using Axin antibody or control IgG, followed by IB with FoxM1 and Axin antibodies..


E . Axin siRNA or control siRNA with HA - Ubiquitin were transfected into 293T cells , after 36 hours , cells were treated with MG132 with or without Wnt - 3a ( 50 ng / ml ) for six hours . Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies .

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E. Axin siRNA or control siRNA with HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.


F . Wild type Flag - FoxM1 or S474A mutant was transfected into 293T cells after 36 hours , cells were treated with MG132 for six hours . Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies .

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F. Wild type Flag-FoxM1 or S474A mutant was transfected into 293T cells after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies.


G . HA - Ubiquitin and / or FBXW7 , GSK3β CA , Flag - FoxM1 were co - transfected into 293T cells , after 36 hours , cells were treated with MG132 for six hours . Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies .

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G. HA-Ubiquitin and/or FBXW7, GSK3β CA, Flag-FoxM1 were co-transfected into 293T cells, after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies.


H . FBXW7 siRNA or control siRNA and HA - Ubiquitin were transfected into 293T cells , after 36 hours , cells were treated with MG132 with or without Wnt - 3a ( 50 ng / ml ) for six hours . Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies .

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H. FBXW7 siRNA or control siRNA and HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.


A . 293T cells were transfected with USPs cDNA or control plasmid . After 36 hr of transfection , cells were treated with 50 ng / ml Wnt - 3a for 4 hr . FoxM1 protein levels in the cells were then determined by Western blotting .

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A. 293T cells were transfected with USPs cDNA or control plasmid. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a for 4 hr. FoxM1 protein levels in the cells were then determined by Western blotting.


B . 293T cells were transfected with 1 µg control plasmid or USP5 cDNA in 6 - well plates . After 36 hr of transfection , cells were treated with 100 µg / ml CHX for 0 , 2 , 4 , or 6 hr . FoxM1 protein levels in the cells were then determined by Western blotting .

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B. 293T cells were transfected with 1 µg control plasmid or USP5 cDNA in 6-well plates. After 36 hr of transfection, cells were treated with 100 µg/ml CHX for 0, 2, 4, or 6 hr. FoxM1 protein levels in the cells were then determined by Western blotting.


C . GSK3 - β CA , USP5 , or control plasmid was co - transfected with Flag - FoxM1 into 293T cells . After 36 hr of transfection , cells were treated with 50 ng / ml Wnt - 3a or control PBS for 4 hr . Cell lysates were subjected to IP with USP5 antibody and followed by IB with Flag or USP5 antibody .

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C. GSK3-β CA, USP5, or control plasmid was co-transfected with Flag-FoxM1 into 293T cells. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a or control PBS for 4 hr. Cell lysates were subjected to IP with USP5 antibody and followed by IB with Flag or USP5 antibody.


D . GSK3βCA , USP5 , or control plasmid was co - transfected with Flag - FoxM1 into 293T cells . After 36 hr of transfection , cells were treated with 50 ng / ml Wnt - 3a or control PBS for 6 hr . Cell lysates were subjected to USP5 with Flag antibody and followed by USP5 with Flag or USP5 antibody .

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D. GSK3βCA, USP5, or control plasmid was co-transfected with Flag-FoxM1 into 293T cells. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a or control PBS for 6 hr. Cell lysates were subjected to USP5 with Flag antibody and followed by USP5 with Flag or USP5 antibody.


E . USP5 and Flag - FoxM1 ( WT ) or mutants Flag - FoxM1S474A or Flag - FoxM1S474E plasmids were co - transfected into 293T cells for 36 hr . Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody .

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E. USP5 and Flag-FoxM1 (WT) or mutants Flag-FoxM1S474A or Flag-FoxM1S474E plasmids were co-transfected into 293T cells for 36 hr. Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody.


F . GSK3β CA , USP5 , or control plasmid was co - transfected with Flag - FoxM1 ( WT ) or mutants Flag - FoxM1S474A into 293T cells for 36 hr . Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody .

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F. GSK3β CA, USP5, or control plasmid was co-transfected with Flag-FoxM1 (WT) or mutants Flag-FoxM1S474A into 293T cells for 36 hr. Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody.


G . HA - ubiquitin and Flag - FoxM1 were co - transfected with or without GSK3β CA into 293T cells . After 36 hr , cells were treated with 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with Flag followed by IB with HA or FoxM1 antibody .

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G. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without GSK3β CA into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag followed by IB with HA or FoxM1 antibody.


H . HA - ubiquitin and Flag - FoxM1 were co - transfected with or without siUSP5 into U87 cells . After 36 hr , cells were treated with 50 ng / ml Wnt - 3a and 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with Flag antibody followed by IB with HA or FoxM1 antibody .

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H. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without siUSP5 into U87 cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a and 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag antibody followed by IB with HA or FoxM1 antibody.


A . Expression of FoxM1 and ICAT was examined via IHC staining in 30 GBM and paired adjacent normal tissues . Representative ICAT and FoxM1 expressions are shown in a section of a GBM tumor with adjacent normal brain . Scale bar : 50 µmB . ICAT stainings were scored as 1 - 12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues . Boxes indicate interquartile range . Bars from each box extend to largest and smallest observations . C . FoxM1 stainings were scored as 1 - 12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues . Boxes indicate interquartile range . Bars from each box extend to largest and smallest observations .

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A. Expression of FoxM1 and ICAT was examined via IHC staining in 30 GBM and paired adjacent normal tissues. Representative ICAT and FoxM1 expressions are shown in a section of a GBM tumor with adjacent normal brain. Scale bar: 50 µmB. ICAT stainings were scored as 1-12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues. Boxes indicate interquartile range. Bars from each box extend to largest and smallest observations.C. FoxM1 stainings were scored as 1-12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues. Boxes indicate interquartile range. Bars from each box extend to largest and smallest observations.


D . Increased amounts of FoxM1 plasmids were transfected into 293T cells . After 36 hr , cells were treated with 50 ng / ml Wnt - 3a for 6 hr . Cell lysates were then subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies .

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D. Increased amounts of FoxM1 plasmids were transfected into 293T cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a for 6 hr. Cell lysates were then subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.


E . Increased amounts of ICAT plasmids were transfected into 293T cells . After 36 hr , cells were treated with 50 ng / ml Wnt - 3a for 6 hr . Cell lysates were then subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies .

E. Increased amounts of ICAT plasmids were transfected into 293T cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a for 6 hr. Cell lysates were then subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.


F . FoxM1 siRNA or control siRNA was transfected into U87 cells , lysates were subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies .

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F. FoxM1 siRNA or control siRNA was transfected into U87 cells, lysates were subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.


G . ICAT siRNA or control siRNA was transfected into LN229 cells , lysates were subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies .

G. ICAT siRNA or control siRNA was transfected into LN229 cells, lysates were subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.


H . ChIP analyses of endogenous β - catenin binding at the TCF - binding site of the Axin2 promoter were performed in vector - , FoxM1 - or / and ICAT plasmid - transfected U87 cells . Fold was calculated relative to that in cells transfected with the vector , which was set as 1 .

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H. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in vector-, FoxM1- or/and ICAT plasmid-transfected U87 cells. Fold was calculated relative to that in cells transfected with the vector, which was set as 1.


I . ChIP analyses of endogenous β - catenin binding at the TCF - binding site of the Axin2 promoter were performed in FoxM1 siRNA - , ICAT siRNA - , control siRNA - or combination of FoxM1 siRNA and ICAT siRNA - transfected U87 cells as described in ( H ) .

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I. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in FoxM1 siRNA-, ICAT siRNA-, control siRNA- or combination of FoxM1 siRNA and ICAT siRNA-transfected U87 cells as described in (H).


J . ChIP analyses of endogenous β - catenin binding at the TCF - binding site of the Axin2 promoter were performed in vector - , β - catenin - NLS - , sh - FoxM1 - , FoxM1 - shR - and / or ICAT plasmid - transfected U87 cells as described in ( H ) .

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J. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in vector-, β-catenin-NLS-, sh-FoxM1-, FoxM1-shR- and/or ICAT plasmid-transfected U87 cells as described in (H).


A . FoxM1 or / and ICAT plasmids were co - transfected with Axin - 2 promoter luciferase and TK - Renilla plasmid into U87 cells . Luciferase activity was measured with the Dural Luciferase reporter assay , and relative luciferase activity was normalized to that of the vector group . Values are mean ± SD for triplicate samples .

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A. FoxM1 or/and ICAT plasmids were co-transfected with Axin-2 promoter luciferase and TK-Renilla plasmid into U87 cells. Luciferase activity was measured with the Dural Luciferase reporter assay, and relative luciferase activity was normalized to that of the vector group. Values are mean ± SD for triplicate samples.


B . FoxM1 siRNA # 1 , # 2 or control siRNA were co - transfected to U87 cells with Axin - 2 promoter reporter . Luciferase activity was measured with the Dural Luciferase reporter assay , and relative luciferase activity was normalized to that of the control siRNA group . Values are mean ± SD for triplicate samples .

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B. FoxM1 siRNA#1, #2 or control siRNA were co-transfected to U87 cells with Axin-2 promoter reporter. Luciferase activity was measured with the Dural Luciferase reporter assay, and relative luciferase activity was normalized to that of the control siRNA group. Values are mean ± SD for triplicate samples.


C . ICAT siRNA or control siRNA were co - transfected into LN229 cells with Axin - 2 promoter reporter . Luciferase activity was measured as in ( C ) .

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C. ICAT siRNA or control siRNA were co-transfected into LN229 cells with Axin-2 promoter reporter. Luciferase activity was measured as in (C).


D . FoxM1 or / and ICAT plasmids were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into U87 cells . Luciferase activity of TOP - Flash in the cells was measured as in A . Values are mean ± SD for triplicate samples .

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D. FoxM1 or/and ICAT plasmids were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into U87 cells. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.


E . ICAT , FoxM1 or FoxM1 - ∆ FB ( FoxM1 mutant lacking the forkhead box domain ) plasmid or their combinations were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into LN229 cells . Luciferase activity of TOP - Flash in the cells was measured as in A . Values are mean ± SD for triplicate samples .

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E. ICAT, FoxM1 or FoxM1-∆FB (FoxM1 mutant lacking the forkhead box domain) plasmid or their combinations were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into LN229 cells. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.


F . FoxM1 siRNA and / or ICAT siRNA were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into LN229 cells , and the cells were treated with Wnt3a ( 50 ng / ml ) or control PBS . Luciferase activity of TOP - Flash in the cells was measured as in A . Values are mean ± SD for triplicate samples .

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F. FoxM1 siRNA and/or ICAT siRNA were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into LN229 cells, and the cells were treated with Wnt3a (50 ng/ml) or control PBS. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.


G . Vector - , β - catenin - NLS - , sh - FoxM1 - , FoxM1 - shR - and / or ICAT plasmids were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into U87 cells . Luciferase activity of TOP - Flash in the cells was measured as in ( A ) . Values are mean ± SD triplicate samples .

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G. Vector-, β-catenin-NLS-, sh-FoxM1-, FoxM1-shR- and/or ICAT plasmids were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into U87 cells. Luciferase activity of TOP-Flash in the cells was measured as in (A). Values are mean ± SD triplicate samples.


H . Levels of Axin - 2 and cyclin D1 protein in LN229 cells transfected with FoxM1 siRNA # 1 or # 2 were determined by Western blotting .

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H. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with FoxM1 siRNA#1 or #2 were determined by Western blotting.


I . Levels of Axin - 2 and cyclin D1 protein in LN229 cells transfected with ICAT siRNA were determined by Western blotting .

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I. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with ICAT siRNA were determined by Western blotting.


J . Levels of Axin - 2 and cyclin D1 protein in LN229 cells transfected with FoxM1 or / and ICAT plasmids were determined by Western blotting .

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J. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with FoxM1 or/and ICAT plasmids were determined by Western blotting.


A . A total of 1 × 104 LN229 cells were transfected with control vector , FoxM1 , and / or ICAT plasmids and treated with Wnt3a ( 50 ng / ml ) and were then plated and counted 48 hr after seeding . The proliferation percentage was calculated relative to that in cells transfected with the control vector , which was set as 100 % . Data represent the mean ± SD of three independent experiments .

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A. A total of 1 × 104 LN229 cells were transfected with control vector, FoxM1, and/or ICAT plasmids and treated with Wnt3a (50 ng/ml) and were then plated and counted 48 hr after seeding. The proliferation percentage was calculated relative to that in cells transfected with the control vector, which was set as 100%. Data represent the mean ± SD of three independent experiments.


B . LN229 cells were transfected with vector control , FoxM1 , FoxM1 plus sh - β - catenin or FoxM1 plus ICAT plasmid . The proliferation percentage was determined as described in ( A ) .

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B. LN229 cells were transfected with vector control, FoxM1, FoxM1 plus sh-β-catenin or FoxM1 plus ICAT plasmid. The proliferation percentage was determined as described in (A).


C . LN229 - shFoxM1 cells were transfected with FoxM1 - shR ( siRNA - resistant FoxM1 ) , or FoxM1 - shR plus ICAT plasmid . The cells were then treated with Wnt3a ( 50 ng / ml ) . The proliferation percentage was determined as described in ( A ) .

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C. LN229-shFoxM1 cells were transfected with FoxM1-shR (siRNA-resistant FoxM1), or FoxM1-shR plus ICAT plasmid. The cells were then treated with Wnt3a (50 ng/ml). The proliferation percentage was determined as described in (A).


D . LN229 cells were transfected with USP5 and / or FoxM1 siRNA . The proliferation percentage was determined as described in ( A ) .

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D. LN229 cells were transfected with USP5 and/or FoxM1 siRNA. The proliferation percentage was determined as described in (A).


E . LN229 cells were transfected with USP5 siRNA and / or FoxM1 plasmid . The cells were then treated with Wnt3a ( 50 ng / ml ) . The proliferation percentage was determined as described in ( A ) . Data shown in ( A ) - ( E ) represent the mean ± SD of three independent experiments .

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E. LN229 cells were transfected with USP5 siRNA and/or FoxM1 plasmid. The cells were then treated with Wnt3a (50 ng/ml). The proliferation percentage was determined as described in (A). Data shown in (A)-(E) represent the mean ± SD of three independent experiments.


F - H . Expression of FoxM1 , Cyclin D1 and USP5 proteins was examined via IHC staining in 50 GBM specimens . Staining of FoxM1 , Cyclin D1 , or USP5 was scored as 1 - 12 according to the intensity and percentage . The significance of the correlations was determined by Pearson ' s correlation test .

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F-H. Expression of FoxM1, Cyclin D1 and USP5 proteins was examined via IHC staining in 50 GBM specimens. Staining of FoxM1, Cyclin D1, or USP5 was scored as 1-12 according to the intensity and percentage. The significance of the correlations was determined by Pearson's correlation test.


I - J . Axin2 mRNA levels in the above 50 GBM specimens were examined through RNAscope technology . The staining was scored as 1 - 12 according to the intensity and percentage . The significance of the correlations was determined by Pearson ' s correlation test .

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I-J. Axin2 mRNA levels in the above 50 GBM specimens were examined through RNAscope technology. The staining was scored as 1-12 according to the intensity and percentage. The significance of the correlations was determined by Pearson's correlation test.


A . Cartoon representation of the Dln1 dimer ( PDB 4ZNO ) . Subunit A is colored in gray . The lectin module of subunit B is colored in yellow , whereas the middle and C - terminal moieties of the aerolysin module are colored in hot pink and green , respectively . The pre - stem hairpin is colored in blue . The bound sucrose molecules are shown as light pink sticks . B . The dimeric interface . Residues involved in dimeric interactions are shown as sticks .

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A. Cartoon representation of the Dln1 dimer (PDB 4ZNO). Subunit A is colored in gray. The lectin module of subunit B is colored in yellow, whereas the middle and C-terminal moieties of the aerolysin module are colored in hot pink and green, respectively. The pre-stem hairpin is colored in blue. The bound sucrose molecules are shown as light pink sticks.B. The dimeric interface. Residues involved in dimeric interactions are shown as sticks.


A . A close - up view of the sucrose - binding pocket . Residues hydrogen - bonding with sucrose are shown as yellow sticks , and the sucrose molecule is shown as light pink stick .

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A. A close-up view of the sucrose-binding pocket. Residues hydrogen-bonding with sucrose are shown as yellow sticks, and the sucrose molecule is shown as light pink stick.


B . The glycan binding profile of Dln1 . This profile represents FITC - labeled Dln1 at 200 μg / ml screened against 609 printed glycans on Mammalian Printed Array version 5 . 2 in replicates of six . A list of top 8 hits reacted with a concentration of 200 μg / ml is shown in Appendix Table S1 . The two kinds of Dln1 - binding glycan structures ( No . 214 and 316 ) are illustrated in schematic .

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B. The glycan binding profile of Dln1. This profile represents FITC-labeled Dln1 at 200 μg/ml screened against 609 printed glycans on Mammalian Printed Array version 5.2 in replicates of six. A list of top 8 hits reacted with a concentration of 200 μg/ml is shown in Appendix Table S1. The two kinds of Dln1-binding glycan structures (No. 214 and 316) are illustrated in schematic.


C & amp ; amp ; D . Close - up views of Manα1 - 2Man and Manα1 - 3Man binding sites . The mannobiose molecules are shown as cyan and gray sticks , respectively . The Fo - Fc electron density maps of disaccharides and the disaccharide - binding sites are shown as gray mesh and countered at 3 . 0 . The hydrogen - bonding residues are shown as yellow sticks . Polar interactions with the mannobiose are shown as dashed lines .

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C&amp;D. Close-up views of Manα1-2Man and Manα1-3Man binding sites. The mannobiose molecules are shown as cyan and gray sticks, respectively. The Fo-Fc electron density maps of disaccharides and the disaccharide-binding sites are shown as gray mesh and countered at 3.0 . The hydrogen-bonding residues are shown as yellow sticks. Polar interactions with the mannobiose are shown as dashed lines.


A & amp ; amp ; B . ELISA of Dln1 towards glycosylated gp120 or yeast mannan . The curve represents the average of triplicate detections , using BSA as the control . Points are averages ( ± standard error of the mean ) of triplicate determinations .

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A&amp;B. ELISA of Dln1 towards glycosylated gp120 or yeast mannan. The curve represents the average of triplicate detections, using BSA as the control. Points are averages (±standard error of the mean) of triplicate determinations.


C . Binding of FITC - labeled Dln1 and D135A mutant to saccharomyces cerevisiae cells . The scale bar represents 5 μm .

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C. Binding of FITC-labeled Dln1 and D135A mutant to saccharomyces cerevisiae cells. The scale bar represents 5 μm.


D . Immunoblot analysis of Dln1 and D135A mutant after incubating with S . cerevisiae cells . The seeing bands represent the cell - bound protein after extensive washing the yeast cells . The monomer and oligomer bands are highlighted by the black arrow .

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D. Immunoblot analysis of Dln1 and D135A mutant after incubating with S. cerevisiae cells. The seeing bands represent the cell-bound protein after extensive washing the yeast cells. The monomer and oligomer bands are highlighted by the black arrow.


A . Oligomerization of Dln1 in the presence of S . cerevisiae mannan or liposomes at different pH values . Histograms are averages ( ± standard error of the mean ) of triplicate

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A. Oligomerization of Dln1 in the presence of S. cerevisiae mannan or liposomes at different pH values. Histograms are averages (±standard error of the mean) of triplicate determinations


B & amp ; amp ; C . Dye release velocity from the liposomes in the presence of Dln1 at different concentrations , and various pH values . Liposomes were incubated with Dln1 of different concentrations at pH 5 . 5 , and 1 μM Dln1 at various pH values , respectively .

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B&amp;C. Dye release velocity from the liposomes in the presence of Dln1 at different concentrations, and various pH values. Liposomes were incubated with Dln1 of different concentrations at pH 5.5, and 1 μM Dln1 at various pH values, respectively.


D . Oligomerization of Dln1M5 with or without liposomes at different pH values . All samples for electrophoresis were prepared with boiling at 95 ° C for 5 min , and the comparative gels were processed in parallel . The oligomer band was quantified by Image J software with the average of triplicate detections , using the marker band as a sample processing control . The SDS - PAGE profiles of Dln1 oligomerization were shown in Appendix Fig S4 . Histograms are averages ( ± standard error of the mean ) of triplicate determinations .

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D. Oligomerization of Dln1M5 with or without liposomes at different pH values. All samples for electrophoresis were prepared with boiling at 95°C for 5 min, and the comparative gels were processed in parallel. The oligomer band was quantified by Image J software with the average of triplicate detections, using the marker band as a sample processing control. The SDS-PAGE profiles of Dln1 oligomerization were shown in Appendix Fig S4. Histograms are averages (±standard error of the mean) of triplicate determinations.


E . Liposomes were treated with Dln1M5 or Dln1 of different concentrations at a fixed pH value of 5 . 5 .

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E. Liposomes were treated with Dln1M5 or Dln1 of different concentrations at a fixed pH value of 5.5.


F . The inhibition of dye release in presence of 0 . 5 M Dln1M5 and 25 mM polyethylene glycols ( PEG ) of different molecular weights at a fixed pH value of 5 . 5 . Dye efflux was monitored in a period of 1400 sec . Triton X - 100 at 0 . 1 % was added to completely disrupt liposomes . Dye efflux was measured as a percentage to the maximal release upon addition of Triton X - 100 . Carboxyfluorescein was loaded in the liposomes for the detection of dye efflux .

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F. The inhibition of dye release in presence of 0.5 M Dln1M5 and 25 mM polyethylene glycols (PEG) of different molecular weights at a fixed pH value of 5.5. Dye efflux was monitored in a period of 1400 sec. Triton X-100 at 0.1% was added to completely disrupt liposomes. Dye efflux was measured as a percentage to the maximal release upon addition of Triton X-100. Carboxyfluorescein was loaded in the liposomes for the detection of dye efflux.


A . Negative - stain electron microscopy image of Dln1M5 in presence of liposomes . B . Side views of Dln1M5 oligomers attached to the membrane of liposomes . The inset represents the respective class averages .

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A. Negative-stain electron microscopy image of Dln1M5 in presence of liposomes.B. Side views of Dln1M5 oligomers attached to the membrane of liposomes. The inset represents the respective class averages.


C & amp ; amp ; D . Transmission electron microscope micrograph of dispersed ring - like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer . The inset represents a few 2D class averages showing the octamer pattern from some individual rings .

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C&amp;D. Transmission electron microscope micrograph of dispersed ring-like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer. The inset represents a few 2D class averages showing the octamer pattern from some individual rings.


E . EM picture of a negatively stained 2D crystal of Dln1M5 on the lipid monolayer . The inset represents the computed diffraction pattern Fourier transform of the image . F . Projection map with a P4 symmetry .

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E. EM picture of a negatively stained 2D crystal of Dln1M5 on the lipid monolayer. The inset represents the computed diffraction pattern Fourier transform of the image.F. Projection map with a P4 symmetry.


G . The top , side and cut - open views of the 3D reconstruction of Dln1M5 octamer at 20 Å resolution are shown as gray surface representation . The surface of membrane is indicated by dashed lines . The scale bar is 10 nm .

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G. The top, side and cut-open views of the 3D reconstruction of Dln1M5 octamer at 20 Å resolution are shown as gray surface representation. The surface of membrane is indicated by dashed lines. The scale bar is 10 nm.


A . Top and side views of eight lectin modules docked to the 20 Å EM map . Due to the moderate resolution of the EM map , the flexible aerolysin module cannot be fit in the map . It is important to notice that this model is only a hypothetical fit . Each pair of the lectin modules are colored in yellow and orange , respectively . The β - barrel of γ - hemolysin ( PDB 3B07 ) was used to mimic the β - barrel backbone of Dln1 . The position of cell membrane is indicated by dashed lines .

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A. Top and side views of eight lectin modules docked to the 20 Å EM map. Due to the moderate resolution of the EM map, the flexible aerolysin module cannot be fit in the map. It is important to notice that this model is only a hypothetical fit. Each pair of the lectin modules are colored in yellow and orange, respectively. The β-barrel of γ-hemolysin (PDB 3B07) was used to mimic the β-barrel backbone of Dln1. The position of cell membrane is indicated by dashed lines.


B . B - factor of Dln1 subunit B . The mobility is represented by coil thickness from thin ( low ) to thick ( high ) , and color from blue ( low ) to red ( high ) . Notably , the pre - stem hairpin and distal moiety of aerolysin module are particularly mobile .

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B. B-factor of Dln1 subunit B. The mobility is represented by coil thickness from thin (low) to thick (high), and color from blue (low) to red (high). Notably, the pre-stem hairpin and distal moiety of aerolysin module are particularly mobile.


C . Position of Cα atoms along MD simulations of apo - form ( red ) and the pre - stem hairpin extraction form ( blue ) are shown with the lectin modules superimposed ( 200 structures for each ) .

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C. Position of Cα atoms along MD simulations of apo-form (red) and the pre-stem hairpin extraction form (blue) are shown with the lectin modules superimposed (200 structures for each).


( A ) Bacterially purified 6xHis - rRv2626c and analyzed by Coomassie blue staining ( left ) or immuno blotting ( IB ) with αHis ( right ) .

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(A) Bacterially purified 6xHis-rRv2626c and analyzed by Coomassie blue staining (left) or immuno blotting (IB) with αHis (right).


( B ) BMDMs were incubated with rRv2626c for the indicated times and then cell viability measured with MTT assay .

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(B) BMDMs were incubated with rRv2626c for the indicated times and then cell viability measured with MTT assay.


( C ) Fluorescence confocal images showing in BMDMs treated with Rv2626c ( 2 . 5 μg / ml ) for 30 min , fixed , immunostained with antibodies for His ( Alexa 488 ) and DAPI . Scale bar , 10 μm .

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(C) Fluorescence confocal images showing in BMDMs treated with Rv2626c (2.5 μg/ml) for 30 min, fixed, immunostained with antibodies for His (Alexa 488) and DAPI. Scale bar, 10 μm.


( D and E ) BMDMs from WT mice were treated with LPS ( 100 ng / ml ) or rRv2626c for indicated times ( D ) . BMDMs from WT , TLR2 - / - , TLR4 - / - , MyD88 - / - , TRIF - / - , IRAK1 - / - , TRAF6 - / - , TBK1 - / - mice were treated with 2 . 5 μg / ml rRv2626c or rVector for 18 h ( E ) . Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA .

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(D and E) BMDMs from WT mice were treated with LPS (100 ng/ml) or rRv2626c for indicated times (D). BMDMs from WT, TLR2-/-, TLR4-/-, MyD88-/-, TRIF-/-, IRAK1-/-, TRAF6-/-, TBK1-/- mice were treated with 2.5 μg/ml rRv2626c or rVector for 18 h (E). Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.


( F ) BMDMs were treated with 2 . 5 μg / ml rRv2626c for 1 h , harvested and then subjected to IB for αIκBα and His . αActin was used as a loading control .

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(F) BMDMs were treated with 2.5 μg/ml rRv2626c for 1 h, harvested and then subjected to IB for αIκBα and His. αActin was used as a loading control.


BMDMs were pretreated with 2 . 5 μg / ml rRv2626c or rVector for 1 h , and stimulated with 100 ng / ml LPS ( A for indicated times . ( A Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA .

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BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml LPS (A for indicated times. (A Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.


BMDMs were pretreated with 2 . 5 μg / ml rRv2626c or rVector for 1 h , and stimulated with 100 ng / ml Pam2CSK4 ( B ) B ) Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA .

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BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml Pam2CSK4 (B) B) Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.