FIG. 7.

ASB3 induces TNF-R2 proteasome-dependent degradation. (A) 293 cells were cotransfected with TNF-R2 (1 μg) and 1.5 μg c-Myc-ASB3 or c-Myc-ASB3ΔSB for 48 h, followed by a CHX (25 μg/ml) chase for the indicated times. Whole-cell extracts were immunoblotted with anti-TNF-R2 (top panel), anti-c-Myc (middle panel), and anti-TRAF2 (low panel). (B) 293 cells were cotransfected with TNF-R2 and the empty pcDNA vector (EV), c-Myc-ASB3, or c-Myc-ASB3ΔSB, followed by a CHX chase in the presence or absence of MG132 (10 μM) or NH₄Cl (15 mM). TNF-R2 and ASB3 proteins were detected as described for panel A. (C) 293 cells were cotransfected with wild-type TNF-R2 or TNF-R2 lysine mutants and with either an empty vector or ASB3 and ubiquitin for 48 h, followed by a CHX chase (25 μg/ml) for an additional 5 h. Cells were either left untreated or treated with TNF-α (10 ng/ml) for 30 min. TNF-R2 and ASB3 protein levels were immunoblotted with anti-TNF-R2 or anti-c-Myc as described above. The right panel shows the TNF-R2 protein levels prior to the CHX chase. (D) Inhibition of ASB3 and Elongin-C expression by ASB3 and Elongin-C siRNAs. 293 cells were transfected with a siRNA directed against green fluorescent protein (GFP; control siRNA), ASB3, Elongin-C, or both ASB3 and Elongin-C. The levels of TNF-R2 proteins (top) and β-tubulin (bottom) were analyzed by immunoblotting with anti-TNF-R2 and anti-β-tubulin, respectively. Elongin-C, asb3, and β-actin mRNA levels were measured by RT-PCR.