Fig. 3. IRS-1 expression and tyrosine phosphorylation. A, nonmalignant and malignant mammary cell lines were grown in complete growth medium and were lysed with RIPA buffer. One mg of lysate protein was immunoprecipitated (IP) with anti-IRS-1, resolved by SDS-PAGE, and immunoblotted (IB) with anti-IRS-1. B, cells were grown to confluence, serum-starved overnight, and incubated for 10 min with or without IGF-I. Prior to lysis, each cell line was treated for 15 min with 40 µM phenylarsine oxide (phosphatase inhibitor) to inhibit phosphatases. One mg of RIPA-extracted lysate protein was immunoprecipitated with anti-IRS-1, resolved by SDS-PAGE, and immunoblotted with anti-phosphotyrosine (anti-pTyr). The positions of the molecular weight markers are indicated. *, nonmalignant.