GSK3β modulates the ACBD5–VAPB interaction via S269. (A) GSK3β (K85A) was coexpressed with Myc-ACBD5 WT or S269A in COS-7 cells. Myc-ACBD5 was immunoprecipitated, and endogenous bound VAPB was detected by immunoblotting using Myc/VAPB antibodies. VAPB (IP fraction) was normalized against total VAPB (input) and Myc-ACBD5 (IP fraction). Phosphorylation of ACBD5 S269 (pS269) was detected by immunoblotting using ACBD5 pS269/Myc antibodies. GSK3β catalytic (in)activity was confirmed using GSK3β/GSK3β pY216/β-catenin antibodies. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test (n = 4). (B) Recombinant His-ACBD5 was incubated in the absence or presence of recombinant GST-GSK3β. Phosphorylation of ACBD5 at S269 was examined by immunoblotting using ACBD5 pS269/ACBD5 control antibodies. (C) Representative electron micrographs of peroxisome–ER contacts in COS-7 cells transfected with a catalytically inactive GSK3β (GSK3β K85A) or GSK3β WT. (D) Assessment of the mean peroxisome membrane surface in direct contact with the ER. Quantitative analysis of the mean population of peroxisomes associated with the ER. Data were analyzed by a two-tailed unpaired t test. Results of four grids per condition. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Bars: 200 nm. Source data are available for this figure: SourceData F7.