Relationship of Gab2 and Lyn. Gab2 is a Lyn substrate. (A) Inhibition of Jak2 tyrosine phosphorylation by AG490, but not by the Src inhibitor PD173. Ba/F3GR cells were first incubated with varying concentrations of Jak2 inhibitor (AG490) or Src kinase inhibitor (PD173), then stimulated with 100 ng/mL G-CSF for 10 minutes. Jak2 was immunoprecipitated and then blotted sequentially with antiphosphotyrosine mAb 4G10 and anti-Jak2. (B) Ba/F3 G-CSFR cells were first incubated with Src kinase inhibitors (10 μM PD173 or 10 μM PP1), Jak2 inhibitor (100 μM AG490), or PI3-kinase inhibitor (10 μM wortmannin or 10 μM LY294003) for 60 minutes, then stimulated with 100 ng/mL G-CSF for 10 minutes. Gab2 tyrosine phosphorylation was then assayed. (C) Dose inhibition of Src inhibitor PD173 on G-CSF-induced tyrosine phosphorylation of Gab2. (D) Lyn-deficient DT40 GR cells without Gab2 tyrosine phosphorylation. DT40 GR and Lyn-deficient DT40 GR were studied for tyrosine phosphorylation of immunoprecipitated Gab2 following stimulation with 100 ng/mL G-CSF.