Defective Rap1 activation by KC in β-arr 2–depleted cells. (A,B) Effect of either β-arr isoform depletion on Rap1 activation by KC. (A) Cells were stimulated with 0.5 μg/mL KC for 1 minute. Rap1-GTP was determined in pull down assays with the effector protein GST-RalGDS-RBD and detected by immunoblotting with a Rap1-specific antibody. Total Rap1 levels in whole cell lysates were used as loading control. (B) Quantitative analysis of KC-induced Rap1 activation in control and β-arr–depleted CXCR2⁺ RBL-2H3 cells. Rap1-GTP levels were quantified by densitometry and normalized both to the total Rap1 and to the GST-RalGDS-RBD signals. Values represent the percentage of normalized Rap1-GTP levels relative to control siRNA-transfected cells. Data are expressed as mean ± SEM of at least 8 independent experiments (*P < .001 for β-arr 2–depleted vs control RBL-2H3 cells). (C) Subcellular localization of Rap1 in RBL-2H3 cells. RBL-2H3 cells were transiently transfected with CXCR2 along with β-arr 2–DsRed.M1. Cells were left unstimulated (0 minutes) or stimulated with 0.5 μg/mL KC for 1 minute, fixed, permeabilized, stained, and analyzed by confocal microscopy. Boxed inserts i-iv represent areas enlarged in images v-viii. Arrows indicate colocalization of Rap1, β-arr 2, and F-actin in ruffling membranes. Scale bars represent 10 μm. Representative images from one of 4 separate experiments are shown. (D-E) Additive effects of PLC inhibition and β-arr 2 depletion on Rap1 activation. CXCR2⁺ RBL-2H3 transfectants were pretreated for 30 minutes at 37°C with either vehicle control or increasing concentrations of U-73122 (0.5 and 1 μM) before addition of 0.5 μg/mL KC for 1 minute. Rap1-GTP was determined in pull down assays and quantified as described above. Quantitated values represent the percentage of normalized Rap1-GTP levels relative to vehicle-treated control cells stimulated with KC. Results are expressed as mean ± SEM of 4 independent experiments (*P < .05; **P < .005).