Fig. 4. Hsp72 interacts directly with JNK1 in vitro. (A) Purified His-Hsp72 protein was added to recombinant GST control, GST–JNK1 or GST–SEK1 protein immobilized on glutathione–agarose beads. Bound proteins were extensively washed, eluted, separated by SDS–PAGE on a 10% polyacrylamide gel and then analyzed by immunoblot using mouse anti-Hsp72 monoclonal antibody. (B) GST control, GST–JNK1 or GST–SEK1 protein was added to His-Hsp72 immobilized on Ni²⁺-NTA–agarose beads. Bead-bound proteins were then washed and eluted from the beads. The eluted proteins were subjected to SDS–PAGE on a 10% polyacrylamide gel, and detected by immunoblotting using mouse anti-GST monoclonal antibody. (C) NIH 3T3 cell lysates were applied to His-Hsp72 that was immobilized on Ni²⁺–agarose beads. Bound proteins were extensively washed and eluted from the beads. The eluted proteins were separated by SDS–PAGE and then analyzed by immunoblotting probed with mouse monoclonal anti-JNK1, rabbit polyclonal anti-ERK2 or mouse monoclonal anti-p38 antibody.