Figure 4
Treatment of cells with GCDA disrupts Mule/Mcl-1 interaction and prolongs the half-life of Mcl-1. (A) HepG2 cells expressing high levels of endogenous Mcl-1 were treated with 100 μg/ml cycloheximide 5 min prior to starting the indicated time course (i.e. 0 h, 0.5 h, 1 h, 2 h, 3 h, 6 h) in the absence or presence of GCDA (100 μM). Mcl-1 was analyzed by Western blot. (B) HepG2 cells were metabolically labeled with 35S-methionine. A classic pulse-chase experiment with the same time course as above (i.e. 0 h, 0.5 h, 1 h, 2 h, 3 h, 6 h) was carried out. The half-life of Mcl-1 was determined by electronic autoradiography. (C) HepG2 cells were treated with increasing concentrations of GCDA. A co-immunoprecipitation was carried out using an Mcl-1 antibody. The Mcl-1-associated Mule and Mcl-1 were analyzed by Western blot.