Time course for G-CSF-induced activation of Lyn, Akt, and ERK1/2. Ba/F3GR cells were stimulated with or without 100 ng/mL G-CSF for indicated time periods, then whole-cell lysates were prepared. (A) Lyn activation. Immunoblotting (IB) was performed using anti-phospho-Src Y416 antibody, which detects the activated state of Lyn, and anti-phospho-Lyn Y507, which detects the nonactivated state of Lyn. The blot was stripped and reprobed with antiactin antibody to demonstrate comparable levels of protein loaded in respective lanes. (B) Akt activation. Immunoblotting was performed using anti-phospho-Akt S473 antibody and reprobed with anti-Akt antibody. (C) ERK1/2 activation. Immunoblotting was performed using anti-phospho-ERK1/2 2T202/Y204 antibody and reprobed with antiactin antibody. Comparable results were observed in 4 independent experiments.