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Fig. 1. Detection of phosphorylation of Src at S17. (A) Limited V8 proteolysis of Flag-Src. A diagram of the published digestion pattern for Src is shown (Collett et al., 1979; Roth et al., 1983). The migrations of the fragments a-d are provided in the gel below. Hek293 cells were transfected with Flag-Src and treated with forskolin for 10 minutes or left untreated. Flag-Src was immunoprecipitated and subjected to limited V8 proteolysis as described in Materials and Methods. SDS-PAGE gel pieces were overlaid with the following amounts of V8 protease per well: 1, 0 µg; 2, 0.005 µg; 3, 0.02 µg; 4, 0.05 µg; 5, 0.1 µg. Phosphorylation of Flag-Src at S17 was visualized by immunoblotting with PKA substrate antibody. The migration of protein standards is shown on the left of the blot (kD). The two smallest detectable fragments represent the 16 kD (d) and 18 kD (c) N-terminal fragments, which are circled in both panels. (B) cAMP and isoproterenol induce phosphorylation of endogenous Src in Hek293 cells. Hek293 cells were treated with forskolin/IBMX (F/I) or Isoproterenol (Iso) for the times indicated. Phosphorylation at S17 (pS17) was detected by western blotting with PSAb following immunoprecipitation of endogenous Src. Total Src is shown as a loading control (lower panels). (C) cAMP-induced phosphorylation of endogenous Src in multiple cell types. CHO, PC12 and AtT-20 cells were treated with forskolin/IBMX (F/I) for the times indicated. Phosphorylation at S17 (pS17) was detected following Src immunoprecipitation by western blotting with PSAb. Total Src was detected as a loading control (lower panels).
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