Figure S3. ACBD4 phosphomimetic mutants... | Rockefeller University Press

$ACBD4 phosphomimetic mutants increase VAPB interaction.(A) Subcellular localization of ACBD4/5 constructs. COS-7 cells transfected with Myc-ACBD5 WT, mFFAT, S269E, S259A/S261A/S263A, or ΔTMD; or FLAG-ACBD4 WT, S183E, or S166A/S169A/S171A, were immunolabeled with PEX14 (peroxisomal marker) and Myc/FLAG antibodies. Bars: 10 µm (main), 2.5 µm (insets). (B and C) ACBD4 constructs with nonphosphorylatable (S → A) and phosphomimetic (S → E) residues upstream (S166/S169/S171) or within the FFAT core (S183) were generated and expressed in COS-7 cells. The FLAG-tagged proteins were immunoprecipitated, and endogenous bound VAPB was detected by immunoblotting using FLAG/VAPB antibodies. (B) Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test (n = 5). Total VAPB (IP fraction) was normalized against total VAPB (input) and FLAG-ACBD4 (IP fraction). (D) Quantification of peroxisome morphology in Myc-ACBD5 (S269E)- or FLAG-ACBD4 (S183E)-transfected COS-7 cells. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. n = 400 per condition, from four replicates. *, P < 0.05; ****, P < 0.0001. TMD, transmembrane domain. Source data are available for this figure: SourceData FS3.$

Figure S3.

ACBD4 phosphomimetic mutants increase VAPB interaction. (A) Subcellular localization of ACBD4/5 constructs. COS-7 cells transfected with Myc-ACBD5 WT, mFFAT, S269E, S259A/S261A/S263A, or ΔTMD; or FLAG-ACBD4 WT, S183E, or S166A/S169A/S171A, were immunolabeled with PEX14 (peroxisomal marker) and Myc/FLAG antibodies. Bars: 10 µm (main), 2.5 µm (insets). (B and C) ACBD4 constructs with nonphosphorylatable (S → A) and phosphomimetic (S → E) residues upstream (S166/S169/S171) or within the FFAT core (S183) were generated and expressed in COS-7 cells. The FLAG-tagged proteins were immunoprecipitated, and endogenous bound VAPB was detected by immunoblotting using FLAG/VAPB antibodies. (B) Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test (n = 5). Total VAPB (IP fraction) was normalized against total VAPB (input) and FLAG-ACBD4 (IP fraction). (D) Quantification of peroxisome morphology in Myc-ACBD5 (S269E)- or FLAG-ACBD4 (S183E)-transfected COS-7 cells. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. n = 400 per condition, from four replicates. *, P < 0.05; ****, P < 0.0001. TMD, transmembrane domain. Source data are available for this figure: SourceData FS3.

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