Figure 6

The nuclear localization of the α isoform of heat shock protein 90 (Hsp90α) is enhanced by treatment of cells with PRIMA-1. MDA-231 cells treated with PRIMA-1 were subjected to nuclear isolation. A fraction of the isolated nuclear pellet was fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 and incubated overnight with anti-p53 and anti-Hsp90α monoclonal antibodies. Nuclear fractions were then immunostained with a secondary antibody conjugated with Oregon green to detect p53 (green) and a secondary antibody conjugated with Texas red to detect Hsp90α (red) before detection by fluorescence microscopy. Nuclei were stained with 4',6-diamidino-2-phenylindole (blue). Normal mouse immunoglobulin G was used as a negative control (data not shown). Arrows mark nuclear staining of Hsp90α. Scale bar, 5 μm.