Fig. 4.

Hsp90 is expressed in AR42J cells and associates with cytosolic rab-GDI-1. A. Hsp90 expression and localization in AR42J cells in the absence (Control) or presence (Dex) of Dex was examined. Cytosol (C) and membrane (M) fractions were prepared from control and Dex-treated cells and immunoblotted for Hsp90 as described in Methods. B. Immunoprecipitation of Rab-GDI-1 from AR42J cell cytosol. Cytosol was prepared from four T-75 flasks of Dex-treated AR42J cells. The cytosol was incubated overnight with a 30 μg GDI-1-specific antibody (GDI ab-right lane) or rabbit IgG (IgG-middle lane), followed by a three hr incubation with protein G-agarose beads. The beads were washed and boiled into SDS sample buffer as described in Methods. The cytosol (left lane) and immunoprecipitates were immunoblotted for Hsp90 and rab-GDI. The asterisk in the GDI-1 blot is a non-specific band and most likely represents amylase.