Fig. 2. Siah2 mediates TRAF2 degradation in vivo. (A) HeLa cells were transfected with Flag-TRAF2, green fluorescent protein (GFP) and Flag-Siah1 or Flag-Siah2. After 24 h, the cells were mock treated or treated with MG132 (40 µM) for 4 h. Cells were harvested, washed and pellets were solubilized with RIPA buffer, and steady-state levels of Flag-TRAF2, Flag-Siah2 and Flag-Siah1 were detected with anti-Flag antibody; the same membrane was reprobed with anti-GFP antibody. (B) Steady-state levels of exogenously expressed TRAF2 were monitored following expression of Siah2 in the presence of either proteasome inhibitor (MG132, 40 µM) or caspase inhibitor (Z-VAD-FMK; 50 µM). HeLa cells were transfected with Flag-TRAF2 and Flag-Siah2, and 24 h later cells were mock treated or treated with MG132 or Z-VAD for 4 h. Cells were then harvested and solubilized with RIPA buffer. Total TRAF2 and Siah2 levels were detected with anti-Flag antibodies. The membrane was reprobed with antibodies to β-actin. (C) HeLa cells were transfected with Flag-TRAF2, Flag-Siah2 and/or RING domain-mutated Flag-Siah2-Rm (H99A/C102A). After 24 h, the cells were mock treated or treated with MG132 (40 µM) for 4 h, and steady-state levels of Flag-TRAF2, Flag-Siah2 and Flag-Siah2-Rm were detected with anti-Flag antibody. (D) HeLa cells were transfected with Flag-TRAF2 and/or Flag-Siah2. After 24 h, cells were harvested, and steady-state levels of transfected (Flag-TRAF2) and endogenous TRAF2 were detected with anti-TRAF2 antibody (Santa Cruz; A20). The same membrane was probed with anti-α-tubulin antibody.