Inhibition of Hsp90 influences AKT degradation and intracellular accumulation of rNadA.

A: Chang cells were incubated for 1, 4, 24 and 48 hours with either 17-AAG or FITC-GA at two different concentrations. At the end of each period cells were washed, trypsinized and RIPA-buffer total lysate prepared. Proteins from each extract were separated on NuSieve gel, blotted, and AKT or Actin (loading control) were detected using the respective primary antibodies. B: Chang cells were pre-treated with 0.01% saponin in PBS for 30 seconds at room temperature, washed three times with PBS and then handled as described in A. C: Chang cells were pre-treated for 1 hour with either vehicle (upper panels), 10 µM 17-AAG (middle panel) or 10 µM FITC-GA (lower panel), and then incubated with 200 µg/ml rNadA at 37°C for 1 (left panels) or 4 hours (right panels). Cells were then fixed, permeabilized and stained for rNadA. The drugs were present during the entire incubation period. IF intensity was calculated as mean ± s.e.m in two independent experiments, each assessing 10–15 cells and expressed as Arbitrary Units (A.U.). Scale bar 10 µm.