Effects of celastrol on HSP90's ATPase activity, HSP70 expression and the HSP90-Cdc37-Cdks complex. A: Celastrol inhibits the activity of ATPase. Co-immunoprecipition of HSP90 was performed on untreated cells, and the beads-bound immunoprecipites were separated into three equal portions before incubation with celastrol, 17-AAG, or DMSO. ATPase activity was determined as the decrease of the absorbance at 340 nm, detailed in Methods. B: Celastrol induces the increase of HSP70. Cells treated with 600 nM celastrol for 1 d and HSP70 levels were deteceted by flow cytometry, as detailed in Methods. X-axis shows channel number and Y-axis shows cell number. C: Celastrol disrupts the HSP90/Cdc37/Cdks complexes. Cells treated with 600 nM celastrol for 1 d, then were used for immunoprecipitation by anti-HSP90 (H9010), detailed as in Methods. The left column (input) displays the detection based on total proteins of cells, while the right column (IP) shows detection of immunoprecipitation using the anti-HSP90 (H9010) antibody.