fMLP-stimulated activation of Ras, p42/p44 MAPKs, PKB and PIP3 accumulation is reduced in RasGRP4kof/kof mouse neutrophils. Neutrophils were prepared from either RasGRP4kof/kof or RasGRP4+/+ mice and their responses to fMLP (10 μM) in a range of assays was compared. (A) The lower part shows representative immunoblots measuring fMLP-, or vehicle- (plotted at 0 s in the graphs), stimulated Ras activation (by RafRBD pull down) and p42/p44 MAPK activation, with anti-phospho-T202/Y204 antibodies, and the amount of β-actin, as a loading control, in aliquots of the lysates from which the pull downs were performed. The upper part presents quantification of data from three independent experiments, like those shown in the lower part, showing activation of Ras. Data are presented as means±s.e., n=3 and are expressed as % of the samples stimulated for 1 min (100%), normalized to their respective loading control. (B) Quantification of p42/p44 MAPK phosphorylation from three separate experiments, like those shown in the lower part of (A) is shown. Data are presented as means±s.e., n=3 and are expressed as % of the samples stimulated for 1 min (100%), normalized to their respective loading controls. (C) Quantification of stearoyl/arachidonoyl-PIP3, as a ratio with the recovered PIP3-ISD, in fMLP- (10 μM) or vehicle (plotted at 0 s) -stimulated neutrophils, from three independent experiments is shown. Data are presented as means±s.e., n=6 (D) The upper part shows a representative immunoblot measuring fMLP-, or vehicle- (plotted at 0 s in the graph), stimulated activation of PKB, with anti-phospho-S473-PKB antibodies, aliquots of lysates were from the experiment shown in (A). The lower part shows quantification of phosphorylation of S473-PKB from three independent experiments for RasGRP4+/+ and two independent experiments for RasGRP4kof/kof, like that shown in the upper part, as means±s.e., n=3 for RasGRP4+/+ or as means±s.d., n=2 for RasGRP4kof/kof and are expressed as a % of the samples stimulated for 1 min (100%), normalized via their respective loading controls. Figure source data can be found with the Supplementary data.