Figure 3.

CIIA physically interacts with ASK1. (A) ³⁵S-Labeled ASK1 was produced by in vitro translation and incubated at 4°C for 3 h with GST-fused CIIA variants immobilized on glutathione-agarose beads. The bead-bound proteins were eluted and analyzed by SDS-PAGE and autoradiography. A lower part of the polyacrylamide gel was cut and stained with Coomassie brilliant blue to show the amount of GST-fused CIIA variants bound on the beads. (B) Binding of in vitro–translated ³⁵S-labeled ASK1 variants to GST-CIIA was examined as in A. In A and B, the input ³⁵S-labeled proteins (10%) were also shown. (C) The soluble fraction of mouse brain tissue or MEF homogenates was precleared with rabbit preimmune IgG and subjected to immunoprecipitation (IP) with rabbit anti-CIIA antibody, or rabbit preimmune IgG. The resulting precipitates were subjected to SDS-PAGE and analyzed by immunoblotting (IB) with anti-ASK1 antibody. Immunoblotting of cell lysates (5% of total) with anti-ASK1 antibody was also shown. (D) 293T cells were transfected with expression vectors encoding ASK1-Flag and HA-CIIA as indicated. After 48 h of transfection, the cells were untreated or treated with 500 μM H₂O₂ for 1 h. Cell lysates were subjected to immunoprecipitation with anti-HA antibody, and the resulting immunoprecipitates were subjected to immunoblot analysis with anti-Flag antibody. Cell lysates were also subjected to immunoblot analysis with the indicated antibodies. (E) L929 cells were untreated or treated with 500 μM H₂O₂ for indicated time periods. Cell lysates were subjected to immunoprecipitation and the resulting immunoprecipitates were analyzed by immunoblotting as in C. Cell lysates (5% of total) were also subjected to immunoblot analysis with anti-ASK1 or anti-CIIA antibody.