FIG. 2.
Insulin-induced IRS-1 degradation requires the activity of ubiquitin-activating enzyme E1. E36 and ts20 cells overexpressing the human insulin receptor (E36/IR and ts20/IR, respectively) were exposed to nothing or 100 nM insulin for 12 h at 30 or 40°C. Cells were unstimulated or stimulated with insulin for 2 min before being lysed. IRS-1 was immunoprecipitated from lysates with αIRS-1, separated by SDS-7.5% PAGE, and transferred to nitrocellulose membranes. Tyrosine phosphorylation of IRS-1 (IRS-1PY) and protein levels of IRS-1 were detected by immunoblotting analysis with αPY and αIRS-1, respectively. These results are representative of at least two experiments.