Figure 6. GSK3β associates with ACBD5... | Rockefeller University Press

$GSK3β associates with ACBD5 and VAPB.(A) GSK3β (S237E “mFFAT”) was coexpressed with FLAG-VAPB, FLAG-ACBD5 WT/mFFAT/ΔTMD, or control vector (FLAG) in COS-7 cells. FLAG-VAPB/ACBD5 were immunoprecipitated, and bound GSK3β and endogenous VAPB were detected by immunoblotting using FLAG/GSK3β/VAPB antibodies. Asterisk indicates unspecific band (due to reprobing of the blot). (B) GSK3β was coexpressed with FLAG-VAPB (K87D/M89D mMSP) or control vector (FLAG) in COS-7 cells. FLAG-VAPB was immunoprecipitated, and bound GSK3β and endogenous ACBD5 were detected by immunoblotting using FLAG/GSK3β/ACBD5 antibodies. (C) FLAG-ACBD5 constructs with nonphosphorylatable (A) residues in the acidic tract were coexpressed with GSK3β in COS-7 cells. FLAG-ACBD5 was immunoprecipitated, and phosphorylation at S269 (pS269) was detected by immunoblotting using ACBD5 pS269 and ACBD5 control antibodies. Bound GSK3β was detected by immunoblotting using ACBD5 control/GSK3β antibodies. GSK3β (IP fraction) was normalized against total GSK3β (input) and ACBD5 control (IP fraction). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test (n = 3); ***, P < 0.001; ****, P < 0.0001. TMD, transmembrane domain. Source data are available for this figure: SourceData F6.$

Figure 6.

GSK3β associates with ACBD5 and VAPB. (A) GSK3β (S237E “mFFAT”) was coexpressed with FLAG-VAPB, FLAG-ACBD5 WT/mFFAT/ΔTMD, or control vector (FLAG) in COS-7 cells. FLAG-VAPB/ACBD5 were immunoprecipitated, and bound GSK3β and endogenous VAPB were detected by immunoblotting using FLAG/GSK3β/VAPB antibodies. Asterisk indicates unspecific band (due to reprobing of the blot). (B) GSK3β was coexpressed with FLAG-VAPB (K87D/M89D mMSP) or control vector (FLAG) in COS-7 cells. FLAG-VAPB was immunoprecipitated, and bound GSK3β and endogenous ACBD5 were detected by immunoblotting using FLAG/GSK3β/ACBD5 antibodies. (C) FLAG-ACBD5 constructs with nonphosphorylatable (A) residues in the acidic tract were coexpressed with GSK3β in COS-7 cells. FLAG-ACBD5 was immunoprecipitated, and phosphorylation at S269 (pS269) was detected by immunoblotting using ACBD5 pS269 and ACBD5 control antibodies. Bound GSK3β was detected by immunoblotting using ACBD5 control/GSK3β antibodies. GSK3β (IP fraction) was normalized against total GSK3β (input) and ACBD5 control (IP fraction). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test (n = 3); ***, P < 0.001; ****, P < 0.0001. TMD, transmembrane domain. Source data are available for this figure: SourceData F6.

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