Domains of interaction between Bcl-2 and Hsp90β and the effect of GA on this interaction. (A) Schematic structure of Hsp90. (B) Hsp90β and its domains, N, M, and C, were expressed as GST-recombinant proteins in BL21 bacteria and incubated with radiolabeled ³⁵[S]-Met-Bcl-2, as described in “Materials and methods.” Results of 1 of 3 representative experiments are shown. (C) RBL cells were exposed to various concentrations of GA (0-0.5 μM) for 12 hours at 37°C. Bcl-2 was then immunoprecipitated from the cell lysates using anti–Bcl-2 antibody. The interaction of Bcl-2 with Hsp90β was determined by Western blotting using anti-Hsp90, as described in “Materials and methods.” Results of 1 of 3 representative experiments are shown. (D) In vitro dissociation between Hsp90 and Bcl-2. The pull-down assay between GST recombinant proteins (Hsp90 full-length and Hsp90 N-terminus) and ³⁵[S]-Bcl-2 was assessed in the presence of different concentrations of GA (0, 1, 10 μM). Results of 1 of 3 representative experiments are shown.