Bay 61–3606 induced ubiquitin-dependent degradation of Mcl-1 by ERK inactivation.

(A) MCF-7 cells were exposed to MG132 for 1 h and incubated with 2.5 μM Bay 61–3606 for 6 h. Mcl-1 expression was analyzed by Western blotting. (B) MCF-7 cells were transfected with pcDNA-Mcl1-flag (Mcl1-Flag) vector with pcDNA-Ubiquitin-HA (Ub-HA) vector. After 48 h of transfection, Ubiquitin-conjugated Mcl-1 was immunoprecipitated with anti-Flag antibody, and then detected using anti-HA antibody (upper). Transient expression of exogenous Mcl-1 was confirmed by Western blotting using the Mcl-1 antibody (lower). (C, D) MCF-7 cells were exposed to Bay 61–3606 (2.5 μM) alone (C) or in combination with TRAIL (50 ng/ml) for 6 h (D). The expressions and phosphorylations of Mcl-1 (C) or ERK, and GSK3α/β (D) were determined by Western blotting.