FIGURE 3.

IRS-1 interacts with p62 through its YXXM motif. A, schematic representation of IRS-1 showing the various tyrosine residues. N, N terminus; PH, pleckstrin homology domain; PTB, phosphotyrosine binding domain; C, C terminus. B, CHO/IR cells were expressed with wild-type IRS-1 and the mutants F18, 3YMXM, or YCT along with Myc-tagged p62. The cells were serum-starved and stimulated with insulin (100 nm) for 15 min. p62 was immunoprecipitated (IP) using Myc antibody and Western blotted for IRS-1 and p62. C, CHO/IR cells were transfected with Myc-p62 along with wild-type IRS-1 or 3YF mutant and treated with insulin for 15 min. The co-interaction of p62 and IRS-1 was determined by immunoprecipitation with anti-Myc and Western blotting for anti-IRS-1 and anti-Myc. The lysates were Western blotted with anti-IRS-1 to verify the expression of the constructs. Experiments were replicated three times with similar results.