Agonist-stimulated colocalization of the internalized CXCR2 with EGFP-Rab5 in HEK293 cells and RBL-2H3 cells.

HEK293 cells (A-D) and RBL-2H3 cells (E-F) stably expressing CXCR2 were transiently transfected with plasmids encoding EGFP-Rab5. Cells were treated with 200 nM CXCL8 at 37°C for different times as indicated, then fixed in methanol. Cells were incubated with a mouse monoclonal anti-CXCR2 antibody at room temperature for 30 minutes, followed by incubation with a rhodamine-conjugated antimouse antibody at room temperature for 30 minutes. Representative laser-scanning confocal micrographs from 3 independent experiments demonstrating the distribution of CXCR2 (red), EGFP-Rab5 (green), and colocalization of CXCR2 with EGFP-Rab5 (yellow) are shown. Images were processed using Photoshop software (Adobe, San Jose, CA). Bars, 10 μm.