FIGURE 8.

Depletion of ESCRT-I subunit UBAP1 attenuates Akt activation and signaling promoted by GPCRs and RTKs. A–C, HeLa cells transfected with siRNA directed against ESCRT-I subunit UBAP1 or luciferase (Ctrl) were treated with vehicle (0.1% BSA in PBS), 10 μm norepinephrine (A, norepi), 100 ng/ml EGF (B), or 50 nm insulin (C) for 5 min. Equal amounts of whole cell lysates were analyzed by immunoblotting for the indicated proteins and their phosphorylation status. Representative blots from four independent experiments are shown. D and E, role of GPCRs and RTKs on DEPTOR degradation. HeLa cells were treated with vehicle (VEH), CXCL12, norepinephrine (norepi), EGF, or insulin for 3 h, and DEPTOR levels were determined by immunoblotting. E, quantification of DEPTOR levels. Bars represent the average amount of DEPTOR remaining normalized to actin compared with vehicle (VEH; set to 100%) from five independent experiments. Error bars represent S.E. Data were analyzed by a one-way ANOVA followed by Bonferroni's post hoc test (VEH versus CXCL12 or norepinephrine, p = 0.00072, as indicated by the brackets).