Fig. 2.

RNF152 is involved in TSC2-mediated Rheb ubiquitination. a Rheb ubiquitination was detected in RNF152 knockdown cells. The knockdown efficiency of RNF152 was detected via RT-PCR in Supplementary information, Fig. S2d. b RNF152 enhanced Rheb ubiquitination in vitro. c, d Endogenous(c) and exogenous (d) interaction between Rheb and RNF152 was examined in HEK293T cells with EGF stimulation. e RNF152 could specifically interact with His-tagged Rheb detected by GST pull-down assay. f Pull-down assay was performed to detect the interaction between GST-RNF152 and His-Rheb, and Rheb was loaded with GDP or GTP in advance. g Rheb-K8R displayed lower ubiquitination level than Rheb-WT when co-expressed with RNF152. h TSC2-induced Rheb ubiquitination decreased in RNF152 knockdown HEK293T cells. The knockdown efficiency was detected by RT-PCR in Supplementary information, Fig. S2d. i Ubiquitination of Rheb-WT or Rheb-K8R mutant was detected with or without TSC2 in HEK-293T cells. j Co-IP assay was performed to test the interaction between Rheb and RNF152 after co-expressed with TSC2 or TSC2-3Q mutant. k Ubiquitination of Rheb was detected in TSC2-depleted HEK293T cell line. l The model of Rheb ubiquitination: TSC complex inactivates Rheb in the absence of EGF, which consequently promotes Rheb-GDP to interact with RNF152 followed by subsequent Rheb ubiquitination