G-CSF–induced Gab2 association with Lyn. (A) Ba/F3GR and parental Ba/F3 cells were treated ± 100 ng/mL G-CSF for 10 minutes. Lysates were prepared, and proteins were immunoprecipitated with anti-Gab2 antibody. Blotting was performed with anti-phospho-tyrosine (4G10), Gab2, and Lyn antibodies. The phosphorylated double band protein is Lyn. (B) Coimmunoprecipitation assay showed that Gab2 associated with Lyn each other in BaF3GR cells. WCL indicates whole cell lysate. (C) GST pull-down results showed that SH3 domain of Lyn interacted with Gab2. Lane 1: WCL; lane 2: GST; lane 3: GST-unique domain of Lyn; lane 4: GST-unique-SH3 domain; lane 5: GST-unique-SH3-SH2 domain; lane 6: GST-SH2 domain. (D) Ba/F3GR cells were treated with either scrambled siRNA or siRNA against Gab2. Lysates were harvested at 24 hours and blotted for either total Gab2, phospho-Src Tyr416, and total Lyn. Numeric values are shown from densitometric analysis.