Fig. 1.

TSC complex and EGF signaling promote the ubiquitination of Rheb. a TSC1 and TSC2 promoted Rheb ubiquitination in HEK293T cells. The ubiquitinated proteins were purified under the denature condition via Ni-NTA agarose beads and were analyzed by Western Blotting. b Co-transfection of HA-TSC2, Myc-Rheb, Ub-WT, or Ub-R72A mutants to detect Rheb ubiquitination, Rheb ubiquitination was analyzed as in (a). c, d Rheb ubiquitination was detected in TSC2 knockdown cells. e, f Ubiquitination of Rheb was detected by co-transfection of TSC2-WT, TSC2-3Q mutant (e) or TSC2(400-C) mutant (f) in HEK293T cells. g The linkage-specific ubiquitination of Rheb was examined in HEK293T cells. h, i Ubiquitination of endogenous (h) and exogenous (i) Rheb was detected in HEK293T cells with the treatment of EGF. j Rheb ubiquitination level was detected in lysosome samples enriched from cells with or without EGF treatment. k Ubiquitination of WT, active (S16H) and inactive (D60V, D60K, S20N)) forms of Rheb was analyzed via Ni-NTA