Fig. 2.

Gam1 WT and not Gam1 LL/AA cause ubiquitylation and proteasome-dependent decrease of VHL. (A) Tet-on HEK293T cells were induced (DOX). One hour after induction, MG132 or DMSO was added to the medium. nUb was used as a control for proteasome inhibition. Loading control: Tubulin. (B) Phoenix cells were transfected with HA VHL, Flag Ub, and myc Gam1 (WT or mutant LL/AA), harvested, and lysed in denaturing SDS-lysis buffer. One milligram of crude extract was immunoprecipitated (IP) against HA tag and analyzed by IB. Anti-Flag antibody detected ubiquitinated HA VHL. Loading control: Tubulin.