Co-immunoprecipitation of IRS4 with Asb-4 in HEK293 cells and rat hypothalamus. Flag-IRS4 and Myc-Asb-4 vectors were co-transfected into HEK293 cells. Cell lysates were precipitated with anti-Flag (A) or anti Myc (B) antibody-conjugated agarose, and blotted with either anti-Myc or anti-Flag antibody as indicated. C. Myc-Asb-4 vectors were transfected into HEK293 cells. Cell lysates were precipitated with anti Myc antibody-conjugated agarose and blotted with either anti IRS4 or anti Myc antibody. D. Rat hypothalamic extracts was precipitated with anti IRS4 antibody and blotted with either anti Asb-4 or anti IRS4 antibody. Normal IgGs and protein A beads were used as negative control in every experiment. All co-immunoprecipitation experiments were repeated three times.