Hsp72 inhibits ASK1 homo-oligomerization and the interaction between ASK1 and MKK3. (A) NIH 3T3 cells were transfected for 48 h with the indicated combinations of ASK1-Flag, HA-ASK1, and Hsp72 constructs. Cell lysates were then subjected to immunoprecipitation (IP) with a mouse monoclonal anti-Flag antibody, and the resulting precipitates were subjected to immunoblot (IB) analysis with a mouse monoclonal anti-HA antibody (upper panel). Cell lysates were also directly subjected to immunoblot analysis with an antibody to Flag, to HA, or to Hsp72 (lower panels). (B) NIH 3T3 cells were transfected for 48 h with the indicated combinations of vectors encoding ASK1-Flag, HA-MKK3, and Hsp72. Cell lysates were then subjected to immunoprecipitation with an anti-HA antibody, and the resulting precipitates were subjected to immunoblot analysis with an anti-Flag antibody. Cell lysates were also directly subjected to immunoblot analysis as described for panel A.