Rap1b^−/− neutrophils exhibit enhanced actin protrusions. (A) Kinetics of fMLP-induced F-actin assembly under adhesion. Representative images of WT and Rap1b^−/− neutrophils that were stimulated with fMLP on fibrinogen-coated slides for the indicated time and stained with rhodamine/phalloidin (in red). Bar, 10 µm. Bar graphs show lamellipodium area of 60 cells that are representative of 3 independent experiments. Mean ± SD; **, P < 0.01 using an unpaired Student’s t test. (B) Immunofluorescence analysis of F-actin in WT and Rap1b^−/− neutrophils that were stimulated with fMLP and plated on a fibrinogen-coated surface. Bar, 10 µm. Bar graph represents the percentage of cells with multiple protrusions. Mean ± SD; **, P < 0.01 using an unpaired Student’s t test; n = 3 independent experiments. (C) Kinetics of F-actin polymerization in WT and Rap1b^−/− neutrophils after fMLP stimulation in suspension using flow cytometry with respect to unstimulated WT neutrophil fluorescence (n = 3 independent experiments). (D) Sustained or second phase of F-actin measured as the percentage of phalloidin fluorescence at 120–240 s to the fluorescence of unstimulated WT neutrophils. ***, P < 0.001. Data are mean ± SD representative of three independent experiments performed in duplicate. (E, left) Representative image of time kinetics of fMLP and ionomycin-elicited Ca²⁺ release using FACSDiva software. (right) Kinetics analysis of intracellular Ca²⁺ release in WT (blue) and Rap1b^−/− (red) neutrophils using Ca²⁺-sensitive dyes Fluo-3 in calcium-containing buffer in response to differential concentration of fMLP or ionomycin. Data were analyzed by FlowJo software and are representative of three independent experiments.