Fig. 5. Detection of IGF-I-dependent tyrosine phosphorylation. Null+ cells were either untreated or treated with 100 µM pervanadate (pV) for 15 minutes at 37°C. Then the cells were incubated with IGF-I (100 ng/ml) at 4°C for 45 minutes in suspension culture or in adherent condition as indicated. The cells were lysed in TGH buffer, and the cell extracts were subjected to immunoprecipitation. (A) An aliquot (1 mg) of lysate was immunoprecipitated with PLC-1 antibody and analyzed by western blotting with antiphosphotyrosine. (B) The phosphotyrosine blot was stripped and re-probed with PLC-1 antibody.