Fig. 5. Involvement of Src family kinases in collagen I, but not in EGF- or insulin-induced SHIP2 phosphorylation. (A) IPs were carried out from serum-starved HeLa cells that were adherent (Ad), detached (D) or freshly re-plated (RP) on collagen-I-coated dishes (6 µg/cm²) as indicated. Adherent cells were pre-treated with DMSO (-) or Src inhibitor PD173956 at 1 µM (+) for 60 minutes followed by addition of EGF (50 ng/ml for 5 minutes; E) or IGF-1 (25 ng/ml for 10 minutes; I). Adherent samples without any growth factor treatment (Control) are shown as well. Similarly, re-plating of cells was done in the presence of DMSO (-) or 1 µM Src inhibitor PD173956 (+). Anti-SHIP2 or pre-immune IPs were immunoblotted with anti-phosphotyrosine (-PY) or anti-SHIP2 antibodies. PD173958 and PD173956 yielded similar results. (B) Anti-SHIP2 IPs from 3T3-L1 adipocytes treated with insulin (50 nM for 15 minutes) or vehicle (V) were blotted with anti-phosphotyrosine (-PY). Cells were pre-treated with 1 µM of Src inhibitor compounds, PD173956 (#56) or PD180970 (#70), for 60 minutes prior to insulin treatment. The arrows point to tyrosine-phosphorylated SHIP2.

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