Direct interaction between WT1 and Hsp90. (A) Equal amounts of K562 protein extracts were immunoprecipitated (IP) with agarose-conjugated mouse immunoglobulin G (IgG) or anti-Hsp90 antibodies, and immunoprecipitates were subjected to SDS-PAGE and immunoblotted (IB) for WT1 (top panel) and Hsp90 (bottom panel). Input represents ∼5% of the total protein extract used for immunoprecipitation. (B) Subcellular colocalization of WT1 and Hsp90. K562 cells were stained with DAPI (blue, nuclear stain) and antibodies to WT1 (green) or Hsp90 (red), and confocal images were acquired at 100× magnification. (C) GST pull-down assay. In vitro–translated and ³⁵S-methionine–labeled full-length Hsp90 was incubated with GST or GST-WT1 protein immobilized on glutathione-sepharose beads, and bound WT1 was detected by fluorography (top panel). 20% of the in vitro–translated protein was used for pull-downs. The bottom panel shows the purity of GST-fused proteins on a Coomassie blue-stained SDS-PAGE gel. (D) Dose-dependent binding of Hsp90 to WT1. Increasing amounts of ³⁵S-methionine–labeled Hsp90 were added to GST-WT1, and binding was analyzed by autoradiography.