Hematopoietic Role of a GCSFR/Gab2/Shp2/Lyn pathway. (A) Ba/F3GR cells were transduced with lentiviral vectors containing GFP and either scrambled shRNA (control) or shRNA against murine Gab2 or Shp2. Flow sorting was performed for the GFP⁺ fraction. Results are shown. (B) Knockdown was confirmed by Western blotting for either Gab2 or Shp2 protein. Numerical values are shown from densitometric analysis. C) Lysates from G-CSF–stimulated Ba/F3 cells treated with control shRNA or shRNA to Gab2 or Shp2 were blotted for phospho-Src Tyr416 and total Lyn. Densitometric analysis was performed, showing a dramatic increase in phospho-Src Tyr416 (phospho-Lyn Tyr396) only in control shRNA cells. (D-E) MTT assay of Ba/F3GR cells treated with either IL-3 or G-CSF, demonstrating the functional importance of Gab2/Shp2 in proliferation. (F) Methylcellulose colony culture assays were performed on bone marrow mononuclear cells from Gab2^−/− and their wild-type littermates. Bone marrow mononuclear cells (3 × 10⁴) were seeded in methylcellulose containing different concentrations of G-CSF (0-100 g/mL). Colonies derived from colony-forming unit granulocyte (CFU-G) were scored on day 7. Error bar denotes SE (D-F).