FIGURE 1.

Methylene blue inhibits generation of steroid binding activity but not GR·Hsp90 heterocomplex assembly by reticulocyte lysate. A, the assay is shown. GR stripped of Hsp90 (Str) was incubated with reticulocyte lysate (RL) in the absence or presence of 10 μm geldanamycin (GA). Immunopellets were washed and Western-blotted for GR and Hsp90. Duplicate pellets were incubated with [³H]dexamethasone to assay steroid binding activity. B, methylene blue inhibits generation of GR steroid binding activity. Stripped GRs were reactivated by reticulocyte lysate in the presence of the indicated concentrations of methylene blue. **, different from control at p < 0.01. C, methylene blue does not inhibit GR·Hsp90 heterocomplex assembly. Stripped nonimmune (NI) or immune (I) GR pellets were incubated with reticulocyte lysate in the absence or presence of 30 μm methylene blue. Immunopellets were washed and Western-blotted for GR, Hsp90, and Hsp70. A typical radiogram of a Western blot is shown above the bar graph. GR-specific Hsp90 (solid bars) and Hsp70 (open bars) were determined in six separate experiments and are presented in the graph relative to the zero (0) methylene blue control set at 100%.