Figure 5.

PHLPP1 stabilizes SGT1 by preventing its association with RNF41. A, HEK293T cells were transfected with SFB-tagged SGT1 along with Myc-tagged wild-type (WT) RNF41 or catalytically inactive mutant C34A (C/A), and their interaction was tested by immunoprecipitation (IP) with streptavidin beads followed by immunoblotting using Myc antibody. WB, Western blotting. B, 293T cells were transfected with SFB SGT1 along with either Myc RNF41 wild type (WT) or catalytically inactive mutant (C/A), and SGT1 ubiquitination was evaluated using anti-ubiquitin (Ub) antibody. C, 293T cells were transfected with HA Ub wild type, Ub K0, and Ub K48R and the ubiquitination of SGT1 was detected by immunoblotting with anti-ubiquitin antibody. D, cells were transduced with either control shRNA or PHLPP1 shRNA, and the interaction of RNF41 and SGT1 in these cells was tested by immunoprecipitation as indicated. E, cells were transfected with vector control or Myc-tagged PHLPP1, and the interaction of triple-tagged SFB-RNF41 with endogenous SGT1 in these cells was detected by immunoprecipitation with streptavidin beads followed by immunoblotting with SGT1 antibody.