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. 2019 Jul 1;30(14):1655–1663. doi: 10.1091/mbc.E19-03-0144

FIGURE 3:

FIGURE 3:

ATPase activity of VCP is necessary to modulate the interaction of Shoc2 with HUWE1. (A) Endogenous Shoc2 was immunoprecipitated from 293FT cells transfected with wild-type (WT) or catalytically inactive (QQ) mutant of VCP-GFP using anti-Shoc2 antibody. The immunoprecipitates were analyzed by immunoblotting using anti-GFP and -Shoc2 antibodies. (B) Cos-SR cells (depleted of endogenous Shoc2 and stably expressing Shoc2-tRFP) expressing the QQ mutant of VCP-GFP and GST-PSMC5 were fixed, immunostained for GST, followed by immunofluorescence microscopy. Insets show high-magnification images of the region indicated by white rectangles. Scale bars: 10 μm. (C) 293FT cells were cotransfected with VCP-GFP and GST-Shoc2. Cells were then treated with the vehicle (dimethyl sulfoxide [DMSO]) or 5 µM of CB-5083 for 4 h. GST-Shoc2 immunoprecipitates were analyzed using anti-GST and -GFP antibody antibodies. (D) Endogenous Shoc2 was immunoprecipitated from Cos1 cells treated with vehicle (DMSO) or 5 µM of CB-5083 for 4 h. The immunoprecipitates were analyzed by immunoblotting with using anti-VCP, -HUWE1, RAF-1, Shoc2, and -PSMC5 antibodies. (E) GST-Shoc2 was immunoprecipitated from 293FT cells cotransfected with VCP-GFP and GST-Shoc2 and depleted in HUWE1. The immunoprecipitates were analyzed using anti-GFP and -GST antibodies. The results in each panel are representative of those from three independent experiments.