Mapping of Hsp90–WT1 interacting domains by GST pull-down assay. (A) Schematic diagram of Hsp90 domains on the left, with the expression constructs and their binding to WT1 on the right. GST pull-down assays were performed using bacterially expressed GST-WT1 (1-517 aa) and GST (negative control) and in vitro–translated and ³⁵S-methionine–labeled full-length and deletion mutants of Hsp90. Bound proteins were separated by SDS-PAGE and visualized by fluorography. Bottom panel shows the in vitro–translated Hsp90 proteins. (B) Schematic diagram of WT1 domains on the left, with the expression constructs and their binding to Hsp90 on the right. GST pull-down assays were performed using bacterially expressed GST-Hsp90 (1-282 aa), GST (negative control), and in vitro–translated and ³⁵S-methionine–labeled full-length and deletion mutants of WT1 and analyzed in panel A. A vertical line has been inserted to indicate a repositioned gel lane in the bottom panel.