Fig. 4. (A) Inhibition of integrin ß1 or Src-family kinases diminishes Src(Y416)- and PLC1(Y783)-activating phosphorylations. BE cells were plated on Matrigel and either left untreated (control) or treated with either blocking ß1 integrin (4B4) antibody (10 µg/ml), Src inhibitor PP2 (10 µM) or the control compound PP3 (10 µM) for 8 hours. Cells were recovered from the Matrigel and processed for western blotting using antibodies to phosphorylated or unphosphorylated forms of PLC1 and Src as indicated. An inhibitor of EGF receptor AG1478 (10 µM) did not prevent PLC1 phosphorylation in this type of experiment (not shown). (B) Stimulation of integrin ß1 enhances Src (Y416) and PLC1 (Y783) phosphorylation. Serum-starved BE cells were stimulated with either Mn²⁺ (5 µM) or the activating integrin ß1 antibody (TS2/16 10 µg/ml) for 30 minutes and then lysed and processed for western blotting using antibodies to phosphorylated or unphosphorylated forms of PLC1 and Src as indicated. (C) PLC1, Src and GIT1 co-immunoprecipitation is integrin ß1 dependent. BE cells, either untreated (–) or treated (+) with the integrin ß1 blocking antibody 4B4 (10 µg/ml), were plated on Matrigel for 8 hours, then recovered from Matrigel, lysed and immunoprecipitation (IP) was performed with either PLC1 or Src antibodies as indicated at the top. Subsequent western blotting (Wb) of these samples was performed using different PLC1 or Src antibodies or the GIT1 antibody as indicated on the left. In the same experiment, EGF receptor could not be detected in the immunoprecipitates (not shown). (D). Src-family kinases Src, Fyn and Lck can directly phosphorylate the PLC1(Y783) site. Purified PLC1 was incubated with purified Src-family kinases, indicated at the top, in an in vitro phosphorylation assay for 30 minutes. PLC1(Y783) phosphorylation was analysed using specific antibodies whereas autophosphorylation of Src kinases was verified using general anti-phosphotyrosine antibodies by western blotting (top panels). Time course of Src phosphorylation of PLC1(Y783). Src phosphorylation of PLC1 was analysed using a time-course in vitro phosphorylation assay followed by western blotting. Amount of phosphorylation was quantified by densitometry (bottom panel).