FIG. 6.

Effect of LY294002 on IRS-1 degradation in pre-insulin-activated cells. CHO/IR/IRS-1 cells were exposed to nothing (lanes a and b) or 100 nM insulin for 9 h (lanes c, h, j, and l); to some cells, LY294002 (50 μM) was added after 30 min (lane l), 2 h (lane j), or 5 h (lane h) of insulin exposure. Control cells were exposed to LY294002 alone for 4, 7, or 8.5 h (lanes d to f) or to insulin alone for 30 min (lane k), 2 h (lane i), or 5 h (lane g). At the end of the incubation, cells were stimulated with 100 nM insulin for 2 min (lanes b to l) before being lysed in Laemmli sample buffer containing 0.1 M DTT. Proteins were separated by SDS-6% PAGE and transferred to nitrocellulose membranes. Protein levels and tyrosyl phosphorylation of IRS-1 were detected by immunoblotting analysis with αIRS-1 and αPY, respectively. The results are representative of two experiments.