Enhanced ROS production in cells expressing a truncated G-CSF receptor. (A) Increased ROS production in cells expressing the truncated G-CSF receptor. Stable transfectants of Ba/F3 cells, expressing comparable levels of either wild-type or truncated G-CSF receptor, were labeled with dihydrorhodamine123 and unstimulated or stimulated with 100 ng/mL G-CSF for indicated periods of time. The relative ROS level induced by G-CSF was determined by monitoring the increased fluorescence (means ± SD) of rhodamine123 in the cells. (B) Truncated G-CSF receptor knock-in mice showed enhanced ROS production. Neutrophils from bone marrow of wild-type and GRΔ715 mice were loaded with DHR followed by stimulation with 100 ng/mL G-CSF for 60 minutes (green) or not (purple). One representative is shown in the left panel; quantitative results (means ± SD of 3 independent experiments) are shown in the right panel. (C) G-CSF-induced sustained activation of Lyn in Ba/F3GRprox. Immunoblotting was performed using anti-phospho-Src Y416 antibody, which detects the activated state of Lyn. Blot was stripped and reprobed with antiactin antibody to demonstrate comparable levels of protein loaded in respective lanes. (D) G-CSF-induced activation of Akt in Ba/F3GRprox. Immunoblotting was performed using anti-phospho-Akt S473 antibody, then the blot was stripped and reprobed with anti-Akt antibody to demonstrate comparable levels of protein loaded in respective lanes. (E) G-CSF-induced activation of ERK1/2 in Ba/F3GRprox. Immunoblotting was performed using anti-phospho-ERK1/2 T202T204 antibody, then the blot was stripped and reprobed with antiactin antibody to demonstrate comparable levels of protein loaded in respective lanes.