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.