Figure 5. GSK3β affects the ACBD5–VAPB... | Rockefeller University Press

$GSK3β affects the ACBD5–VAPB interaction.(A) Myc-VAPB was expressed in the absence or presence of GSK3β in COS-7 cells. Myc-VAPB was immunoprecipitated, and endogenous bound ACBD5 and PTPIP51 were detected by immunoblotting using Myc/ACBD5/PTPIP51 antibodies. Results of three independent IPs were quantified. ACBD5/PTPIP51 (IP fraction) was normalized against total ACBD5/PTPIP51 (input) and Myc-VAPB (IP fraction). (B) FLAG-ACBD5 was expressed in HEK293T cells. Cells were treated with 10 µM CHIR (GSK3β inhibitor) or DMSO for 16 h. FLAG-ACBD5 was immunoprecipitated, and endogenous bound VAPB was detected by immunoblotting using FLAG/VAPB antibodies. Inhibition of GSK3β by CHIR was confirmed using GSK3β/GSK3β pY216/β-catenin antibodies (arrowhead indicates GSK3β). VAPB (IP fraction) was normalized against total VAPB (input) and FLAG-ACBD5 (IP fraction). n = 5–8 of three independent IPs. Data were analyzed by a two-tailed unpaired t test; **, P < 0.01; ***, P < 0.001. Source data are available for this figure: SourceData F5.$

Figure 5.

GSK3β affects the ACBD5–VAPB interaction. (A) Myc-VAPB was expressed in the absence or presence of GSK3β in COS-7 cells. Myc-VAPB was immunoprecipitated, and endogenous bound ACBD5 and PTPIP51 were detected by immunoblotting using Myc/ACBD5/PTPIP51 antibodies. Results of three independent IPs were quantified. ACBD5/PTPIP51 (IP fraction) was normalized against total ACBD5/PTPIP51 (input) and Myc-VAPB (IP fraction). (B) FLAG-ACBD5 was expressed in HEK293T cells. Cells were treated with 10 µM CHIR (GSK3β inhibitor) or DMSO for 16 h. FLAG-ACBD5 was immunoprecipitated, and endogenous bound VAPB was detected by immunoblotting using FLAG/VAPB antibodies. Inhibition of GSK3β by CHIR was confirmed using GSK3β/GSK3β pY216/β-catenin antibodies (arrowhead indicates GSK3β). VAPB (IP fraction) was normalized against total VAPB (input) and FLAG-ACBD5 (IP fraction). n = 5–8 of three independent IPs. Data were analyzed by a two-tailed unpaired t test; **, P < 0.01; ***, P < 0.001. Source data are available for this figure: SourceData F5.

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