Fig. 3
From: Ubiquitination of Rheb governs growth factor-induced mTORC1 activation
USP4 promotes Rheb activation through removing the ubiquitin from Rheb. a The endogenous interaction between USP4 and Rheb was analyzed via Co-IP assay. b The interaction between USP4 and Rheb was analyzed via pull-down assay. c, d USP4 reversed the effect of TSC2 (c) and RNF152 (d) on Rheb ubiquitination in HEK293T cells. e Vialinin A blocked the effects of USP4 on Rheb deubiquitination. f Endogenous ubiquitination of Rheb was increased in USP4-depleted HEK293T cells. g USP4 directly removed the ubiquitin from Rheb in vitro. h The interaction between Rheb and USP4 was analyzed under EGF treatment for indicated time periods. i The interaction between Rheb and USP4-WT, USP4-S445A, and USP4-S445D. j The effects of USP4-WT, USP4-S445A, and USP4-S445D on RNF152-mediated ubiquitination of Rheb in HEK293T cells. k, l The activation of Rheb was measured in RNF152 deficient (k) or USP4 deficient MEF cells (l) with EGF treatment for indicated time. RNF152-KO MEF cells were analyzed via genomic DNA PCR in Supplementary information, Fig. S3s. m Vialinin A reduced the activation of endogenous Rheb. n The activation of Rheb was measured in HEK293T cells co-expressing Flag-Rheb with USP4-WT or USP4 mutants, respectively. o The model of Rheb deubiquitination by USP4
