FIG. 1.

IRS-1 is tyrosine phosphorylated and then degraded after IGF-I stimulation. (A) MCF-7 cells were incubated in SFM for 24 h and then stimulated with IGF-I (10 nM) for 15 min and 2 h. Cells were lysed in TNESV buffer, and 300 μg of the resultant protein was used for immunoprecipitation experiments with antibodies to IRS-1. Total cell lysate (lanes 1 to 3 from left) and immunoprecipitated proteins (IP:IRS-1) (lane 4 to 6 from left) were separated by SDS–8% PAGE and immunoblotted with antiphosphotyrosine (PY) or anti-IRS-1 (IRS-1) antibodies. (B) MCF-7 cells were treated as described for panel A, but no immunoprecipitation was performed. (C) MCF-7 cells were incubated with IGF-I (0, 0.1, 1, and 10 nM) for 8 h and lysed, and the resulting lysate was treated as described for panel A. All immunoblots are representative of at least four independent experiments. (D) MCF-7 cells were labeled with [³⁵S]methionine-[³⁵S]cysteine for 1 h, incubated in chase medium with or without IGF-I (5 nM), and then lysed at the indicated time points. Lysates were immunoprecipitated with anti-IRS-1 antibodies and separated by SDS-PAGE, and the gel was then dried. Densitometry was performed with a PhosphorImager. Results are expressed as percentages of the densitometric reading at 0 h. This figure is representative of two independent experiments.