P-Rex1 co-localizes with PIP 3 at the plasma membrane leading to Rac1 activation and LFA-1 clustering. (A-B) Co-localization of P-Rex1 (red), PIP2 (green; A), and PIP3 (green; B) in WT (left) and P-Rex1−/− (right) neutrophils with and without E-selectin stimulation. Displayed are representative cells of 3 independent experiments. (C) Quantitative analysis of co-localization of PIP2 vs P-Rex1 and PIP3 vs P-Rex1, respectively. (D) Co-localization of P-Rex1 (red), PIP3 (green), and LFA-1 (purple) after E-selectin stimulation in WT and P-Rex1−/− neutrophils. Shown are representative images of 3 independent experiments. (E) Co-localization of P-Rex1 (red), PIP3 (green), and Mac-1 (purple) after E-selectin stimulation in WT and P-Rex1−/− neutrophils. Shown are representative images of 3 independent experiments. Scale bars indicate 10 μm. (F) Quantitative analysis of the co-localization of P-Rex1 and PIP3 with Mac-1 or LFA-1. (G) Representative western blots of Rac1 and Rac2 activity without stimulation and after stimulation with E-selectin in WT and P-Rex1−/− neutrophils (n = 3). (H) Densitometric analysis of Rac1 and Rac2 activity assays (n = 3) normalized to stimulated WT cells. Bars are adjusted relative density ± SEM. (I) Flow chamber of neutrophils preincubated with WT and DN Rac1 peptides on ICAM-1 and E-selectin/ICAM-1. (J) Corresponding flow chamber of neutrophils preincubated with WT and DN Rac2 peptides. #P < .05.
