FIGURE 1.

Insulin-dependent interaction between p62 and IRS-1. A, L6 myotubes were serum-starved for 4 h and either left untreated or stimulated with insulin (100 nm) for 15 min at 37 °C. One mg of lysate protein was immunoprecipitated (IP) with IRS-1 antibody and Western blotted with antibodies against IRS-1 or p62, n = 3. The lanes labeled Input indicate crude lysate. B, the above lysates were also immunoprecipitated with anti-p62 and Western blotted with IRS-1 and p62 antibody. C, L6 myotubes (400 μg) stimulated with or without insulin were interacted with an equivalent amount of GST-p62 in a pulldown assay. The association of IRS-1 was determined by Western blotting with IRS-1 and GST antibody. D, CHO/IR cells were transfected with V5-IRS-1 and Myc-p62 and stimulated with or without insulin for 15 min. The cells were lysed, immunoprecipitated with V5 antibody, and immunoblotted with IRS-1 and Myc antibody. Lysates were Western blotted with anti-Myc to assess the expression of the p62 plasmid. Experiments were replicated three times with similar results.