FIG. 2.

IGF-I-evoked phosphorylation of nuclear PLC β1 is blocked by the MEK inhibitor PD98059 and U0126. Quiescent Swiss 3T3 cells were radiolabeled with ³²P and incubated without or with 40 ng of IGF-1/ml for the times indicated. The kinase inhibitor (20 μM PD98059, 20 μM U0126, or 20 μM LY2940022) was added 30 min before IGF-I. Nuclear proteins purified from each of these treated cultures (500 μg) were immunoprecipitated by anti-PLC β1 MAb. Immunoprecipitated complexes were eluted with 50 μl of 1× SDS-PAGE loading buffer. A 25-μl portion of each sample was separated by SDS–8% PAGE and analyzed by either autoradiography (A) or Western blotting using anti-PLC β1 (B).