17-AAG promotes ubiquitination and proteasome-dependent degradation of WT1. (A) K562 cells were treated with 10μM 17-AAG alone, 10μM lactacystin alone or with a combination of both agents for 24 hours, and cell lysates were subjected to Western blot analysis using anti-WT1 and anti–β-actin. (B) K562 cells were treated as above for 8 hours, and proteins extracts were immunoprecipitated (IP) with anti-WT1. The ubiquitination of WT1 was analyzed by Western blotting (IB) with anti-ubiquitin (top panel), and WT1 protein levels were assessed by anti-WT1 (bottom panel).