Functional interactions between KC-activated CXCR2 and β-arrestins. (A-C) Subcellular localization of β-arr 1 and β-arr 2 in RBL-2H3 cells. RBL-2H3 cells were transiently transfected with CXCR2 along with either DsRed.M1 (A), β-arr 1-DsRed.M1 (B), or β-arr 2–DsRed.M1 (C). Cells were left unstimulated (0 minutes) or stimulated with 0.5 μg/mL KC for the indicated times (0.5 minutes and supplemental Figure 4), fixed, permeabilized, stained, and analyzed by confocal microscopy. Scale bars represent 10 μm. Representative images from one of 4 separate experiments are shown. (D-E) Retained KC-induced calcium transients in either β-arr isoform-depleted CXCR2⁺ RBL-2H3 cells. (D) Cells were loaded with the calcium indicator Fura 2-AM. Changes in free intracellular calcium levels induced by stimulation with 0.5 μg/mL KC were measured by fluorimetry. Representative curves from one of 3 experiments are shown. (E) Quantitative analysis of KC-induced intracellular calcium mobilization in control and β-arr–depleted CXCR2⁺ RBL-2H3 cells. Results are expressed as mean ± SEM of 3 independent experiments.