Fig. 4.

GR expressed in HEK cells is associated with some Hsp90αβ heterodimers. (a) Purified hHsp90α or hHsp90β were immunoadsorbed with nonimmune (NI), anti-Hsp90α or anti-Hsp90β antibodies to demonstrate the specificity of immunoprecipitation. (b) Equal amounts of purified hHsp90α and hHsp90β were mixed and incubated for 20 minutes at 30°C in the GR•Hsp90 heterocomplex assembly assay buffer with the ATP regenerating system. The mixture was then immunoprecipitated with nonimmune (NI), anti-Hsp90α, or anti-Hsp90β antibodies, and the immune pellets were blotted for Hsp90α or Hsp90β. (c) GR•hHsp90 heterocomplexes formed in HEK cells. HEK cells were transfected with mouse GR cDNA, and 48 hours later, cell lysates were immunoadsorbed with FiGR anti-GR monoclonal (I) or nonimmune (NI) antibody. The washed pellets are First IP. Complexes were released by competition with a peptide encompassing the FiGR epitope (Input) and immunoadsorbed with anti-Hsp90α, anti-Hsp90β, or nonimmune antibody. The gels were Western blotted for GR, Hsp90α, and Hsp90β.