FIG. 5.

26S proteasome inhibitors block IGF-mediated degradation of IRS-1. (A) MCF-7 cells were incubated in SFM for 24 h; pretreated with DMSO, MG132 (50 μM), or lactacystin (10 μM) for 30 min; and then stimulated with IGF-I (10 nM), or not stimulated, in the presence or absence of inhibitor for a further 24 h. Cells were lysed, and 50 μg of the resultant protein was separated by SDS–8% PAGE and immunoblotted with antiphosphotyrosine (PY) or anti-IRS-1 (IRS-1) antibodies. (B) MCF-7 cells were incubated in SFM for 24 h and then in SFM with or without lactacystin (10 μM) for 30 min. Cells were stimulated with increasing concentrations of IGF-I (0, 0.1, 1, and 10 nM) for 24 h in the presence or absence of lactacystin (10 μM). Cells were lysed, and 50 μg of the resultant protein was separated by SDS–8% PAGE and immunoblotted with antiphosphotyrosine (PY) and anti-IRS-1 (IRS-1) antibodies. This figure is representative of three independent experiments.