Effect of Kit ligand on the interaction between Hsp90 and Bcl-2 in BMMCs. (A) BMMCs underwent growth factor starvation for 2 hours, and then 50 ng/mL Kit ligand was added for various periods of time (0, 30, and 60 minutes). Bcl-2 was immunoprecipitated from the cell lysates by anti–Bcl-2. Its interaction with Hsp90 was determined by Western blotting with anti-Hsp90, as described in “Materials and methods.” Results of 1 of 3 representative experiments are shown. (B) BMMCs were synchronized by deprivation of IL-3 for 4 hours and then grown for more 24 hours in the presence of IL-3. After nuclear staining with PI, the cells were sorted by FACS into G1 and G2 phases, as described in “Materials and methods.” Results of 1 of 2 representative experiments are shown. (C) Bcl-2 was immunoprecipitated from G1 and G2 cell lysates, and the presence of Hsp90 was tested by Western blotting. Results of 1 of 2 representative experiments are shown. (D) BMMCs were treated with 15 μM nocodazole for G2/M arrest, and then Hsp90 was immunoprecipitated. The presence of Bcl-2 in the complex was tested using anti–Bcl-2 antibody. Results of 1 of 3 representative experiments are shown.