Identification of Hsp90β as a Bcl-2–interacting protein in RBL cells. (A) Bcl-2–interacting proteins were isolated by immunoprecipitation of Bcl-2 from RBL-cell lysates using an anti–Bcl-2 polyclonal antibody. The samples were electrophoresed on a 10% SDS polyacrylamide gel and then stained with a staining solution. (B) The bands at approximately 90 kDa were excised, digested with trypsin, and analyzed by mass spectrometry, as shown by the deconvoluted spectrum of the unknown protein. (C) Verification of the Hsp90β interaction with Bcl-2 in RBL cells by coimmunoprecipitation assays. Bcl-2 was immunoprecipitated from RBL-cell lysates, and Hsp90β was detected by Western blotting using a monoclonal anti-Hsp90 antibody. Results of 1 of 3 representative experiments are shown. Reciprocal coimmunoprecipitation: Hsp90 was immunoprecipitated from RBL-cell lysates, and Bcl-2 in complex was detected by a specific antibody. Results of 1 of 3 representative experiments are shown. (D) Hsp90 specifically interacts with Bcl-2 but not with other Bcl-2 family members. After immunoprecipitation of Bcl-2, BclX_L, A1, Mcl-1, and Bax from RBL cells, the interaction of Hsp90β with these proteins was assessed by Western blotting using a monoclonal anti-Hsp90 antibody. Results of 1 of 3 representative experiments are shown.