Figure 3.

Effects of overexpressed Hsp90WT and Hsp90NLS on glucocorticosteroid-inducible MMTV promoter activity. (a) MMTV promoter activity in 34i cells transfected with plasmids bearing the reporter plus Hsp90Mut (●), Hsp90WT (○), or Hsp90NLS (▵) in the presence of increasing concentrations of TA. (Inset) Simian virus 40 (SV40) promoter activity in the presence of Hsp90Mut (white bars), Hsp90WT (hatched bars), or Hsp90NLS (black bars). The mean values (±SEM) of luciferase activity, expressed in relative light units (RLU), are from four experiments with each point in triplicate. (b) Inhibition of MMTV promoter activity by increasing levels of Hsp90WT and NLS. 34i cells transfected with the Hsp90 constructs described for a were stimulated or not with 10⁻⁷ M TA for 6 h at the indicated times after transfection. For each point (in triplicate) of the time course experiment, the values of the reporter activity of Hsp90 Mut (white bars) construct stimulated by TA are represented as 100%; Hsp90WT is represented by hatched bars and Hsp90NLS, by black bars. (c) SDS/PAGE and Western blot analysis. Equal amounts of cell lysates from experiments described for b were analyzed by SDS/PAGE and Western blotting to check the level of expression of chicken Hsp90WT and Hsp90NLS (WT and N, respectively).