FIG. 1.

Hsp72 inhibits H₂O₂-induced ASK1 activation in NIH 3T3 cells. (A) NIH 3T3 cells were transiently transfected for 30 h with the pcDNA3 vector encoding Flag-tagged ASK1. The cells were then either left untreated or exposed to a mild heat shock (43°C for 20 min), incubated for an additional 16 h at 37°C, and either left untreated or treated with 2 mM H₂O₂ for 20 min. Cell lysates were subjected to immunoprecipitation with an anti-Flag antibody, and the resulting precipitates were examined for ASK1 activity by an immunocomplex kinase assay with GST-MKK6(K82A) as a substrate (top panel). The abundances of ASK1-Flag and Hsp72 in cell lysates were also examined by immunoblot analysis (IB) with mouse monoclonal antibodies to the Flag epitope (middle panel) and to Hsp72 (bottom panel). (B) NIH 3T3-neo or NIH 3T3-Hsp72 cells were transiently transfected for 48 h with the ASK1-Flag vector and then incubated in the absence or presence of 2 mM H₂O₂ for 20 min or of 600 mM sorbitol for 30 min. Cell lysates were assayed for ASK1 activity by an immunocomplex kinase assay and subjected to immunoblot analysis as described for panel A. (C) NIH 3T3-neo or NIH 3T3-Hsp72 cells were transiently transfected with an expression vector encoding HA-tagged TAK1 or HA-tagged MEKK1, respectively. After 48 h of transfection, cells were incubated in the absence or presence of 600 mM sorbitol for 30 min. Cell lysates were subjected to immunoprecipitation with an anti-HA antibody, and the resulting precipitates were assayed for TAK1 and MEKK1 activities.