FIG. 4.

M11L interacts directly with Bak. (A) M11L interacts with ectopically expressed huBak. 293T cells were transfected with pcDNA3-M11L and pcDNA3HA-huBak or an empty vector. Coimmunoprecipitations (CO-IP) were performed in 2% CHAPS lysis buffer by using anti-M11L or anti-HA (12CA5); immunoblot (WB) analysis detected the resulting immune complexes with anti-HA (upper left panel) or anti-M11L (lower left panel), respectively. Expression of the transfected plasmids is shown in the panels on the right. (B) M11L interacts with endogenous Bak in infected HOS cells. HOS cells were infected with vMyxlac (MOI, 10), and at 12 h postinfection, immune complexes were pulled down with anti-Bak and immunoblotted with anti-M11L (top). Expression of M11L (center) and endogenous human Bak (bottom) is also shown. (C) M11L binds to Bak in the absence of Bax. DU145 human prostate cancer cells (deficient in Bax) were infected with vMyxlac (MOI, 10) for 12 h. Immune complexes were precipitated with anti-Bak and immunoblotted with anti-M11L (top panel). Immunoblotting of lysates confirmed that M11L was expressed only in infected cells (second panel) and that endogenous Bak was detected in the cells (third panel) but endogenous huBax was not (bottom panel). (D) In vitro-translated M11L can bind directly to huBak. Translated products of unlabeled huBak alone (pcDNA3-huBak [lane 1]) or unlabeled huBak plus ³⁵S-radiolabeled M11L (pcDNA3-M11L [lane 2]) were incubated together with anti-Bak-protein A agarose beads. The unlabeled translated huBak alone was precipitated with anti-Bak (lane 3).