FIG. 3.

Insulin-induced Tyr phosphorylation of IRS-1^7A in Fao cells. (A) Fao cells were infected with Ad-Myc-IRS-1^WT or Ad-Myc-IRS-1^7A at 7 × 10⁷ PFU/ml. After 48 h, the cells were starved in serum-free RPMI medium for 16 h and then treated with insulin for the indicated times. Cell extracts were prepared, and samples (1 mg) were subjected to immunoprecipitation (IP) with anti-Myc (α-Myc) antibodies. Immunocomplexes were resolved by SDS-PAGE (7.5% polyacrylamide) and immunoblotted (IB) with anti-PY (α-PY) antibodies. In parallel, samples (100 μg) of total cell extracts were resolved by SDS-PAGE (7.5% polyacrylamide) and immunoblotted with anti-Myc antibodies. (B) The bands corresponding to anti-PY/total Myc-IRS-1 were quantitated by densitometry, and the results are shown in a bar graph. Results of a representative experiment performed with duplicate samples are shown.