G-CSF treatment activates Lyn by the dephosphorylation of Lyn Tyr507 and phosphorylation of Lyn Tyr396. (A) Ba/F3 parent and Ba/F3GR cells were untreated or treated with 100 ng/mL G-CSF for 10 minutes; the whole cell lysates were prepared followed by immunoprecipitating Jak2, blotting with phospho-tyrosine, or by blotting with phospho-Stat1, Stat3, and Stat5, reprobing with anti-Jak2, Stat1, Stat3, and Stat5 antibodies. (B) Ten million cells were harvested from Ba/F3GR cells for each sample. The whole cell lysates were prepared and immunoprecipitated with anti-Hck, Fyn, Blk, Lyn, Fgr, and Src antibodies, respectively, kinase activity was determined by autophosphorylation in the presence of radio-labeled ATP, and the results showed that Lyn is the predominant Src kinase expressed in G-CSF receptor expressing Ba/F3 cells. (C) After 5 hours of IL-3 starvation, Ba/F3GR cells were stimulated with 100 ng/mL G-CSF for the indicated times and lysates were prepared to be blotted with anti-phospho-Lyn Tyr507 or anti-phospho Src Tyr416 (which detects phospho-Lyn Tyr396). The blot was stripped and reprobed to detect total Lyn. Three independent experiments were performed and representative data are shown (top panel). Densitometric analysis was performed from the 3 independent experiments and relative values are shown (bottom panel). Error bar denotes SE.