AR-42 promotes disassociation of Kit from HSP90 without evidence of HSP90 hyperacetylation. (A) Mast cell lines were treated with 3μM (P815 cells) or 1μM (C2 and BR cells) AR-42 or 1μM 17-AAG for 8 hours. HSP90 was immunoprecipitated from the cell lysates, and the levels of Kit and HSP90 in the immunoprecipitates were determined by Western blot analysis (top panel). Western blotting for Kit and HSP90 was also performed on total cell lysates (50 μg) before immunoprecipitation as a control (bottom panel). (B) P815, C2, and BR cells were treated with increasing concentrations of AR-42 or 1μM 17-AAG for 24 hours. Equal amounts of cell lysates were analyzed by Western blotting to detect induced HSP70, HSP90, and β-actin. (C) Mast cell lines were treated with 3μM (P815 cells) or 1μM (C2 and BR cells) AR-42 or 1μM 17-AAG for 24 hours. Protein lysates were generated and, after 7% SDS-PAGE of 200 μg of total protein, Western blotting was performed for acetyl-lysine. Blots were then stripped and reprobed first for total HSP90 and then for acetyl-tubulin. The top panel demonstrates the lack of any 90-kDa acetylated protein, although the 90-kDa HSP90 protein is evident after Western blotting for this protein (middle panel). (D) Mast cell lines were treated with 3μM (P815 cells) or 1μM (C2 and BR cells) AR-42 or 1μM 17-AAG for 24 hours. Protein lysates were generated and, after 10% SDS-PAGE of 200 μg of total protein, Western blotting was performed for acetyl-lysine. Multiple acetylated proteins are evident on the Western blot, although none corresponds directly to HSP90. All experiments shown in this figure were performed 3 times.