Figure 2.

RASAL3 functions as a RasGAP to regulate Ras signaling in neutrophils. (A, B) Assessment of neutrophil numbers in RASAL-KO and WT after LPS challenge. Mice received i.p. injection of LPS or saline (vehicle control). After 24 h, peripheral blood and bone marrow was collected, and neutrophil population (Ly6G+CD11b+ cells) were analyzed by flow cytometry. Representative flow cytometric plots are shown, along with percentage of neutrophils in the peripheral blood in (A). Bone marrow neutrophil frequency are indicated in (B). (C) Bone marrow (BM) isolated neutrophils were treated with LPS or vehicle control at 37°C for 30 min. Cell lysates were then harvested for Western blot after pull down assays. Active Ras (Ras-GTP) was collected by pull-down. Ras-GTP in the sample and total Ras in whole cell lysate were detected by western blot (WB). A representative image of WB (left) and fold intensities of total Ras (middle) and Ras-GTP (right) in the WB is shown. (D–F) Assessment of activation level of downstream inflammatory signaling factors in neutrophils. Both total and phosphorylated levels of NF-κB p65 (D), p38 MAPK (E) and Akt (F) in cell lysate were detected by ELISA. Data are shown as the mean ± SEM of five samples from one of two independent experiments. Each symbol represents data from one mouse. *p < 0.05, **p < 0.01 and ***p < 0.001.