Agonist-stimulated internalization of CXCR2 in HEK293 cells expressing EGFP-Rab5-Q79L or EGFP-Rab5-S34N.

HEK293 cells stably expressing CXCR2 were transiently transfected with plasmids for EGFP-Rab5-Q79L (A-B) or EGFP-Rab5-S34N (C-D). Cells were treated with 200 nM CXCL8 at 37°C for 30 minutes, then fixed in methanol. Cells were incubated with a mouse monoclonal anti-CXCR2 antibody for 30 minutes, followed by incubation with a rhodamine-conjugated antimouse antibody for 30 minutes. Representative confocal micrographs from 3 independent experiments demonstrating the distribution of CXCR2 (A, C) in cells expressing EGFP-Rab5-Q79L (B) or EGFP-Rab5-S34N (D) are shown. Small arrows indicate the examples of colocalization of CXCR2 with EGFP-Rab5-Q79L (A-B). Large arrows indicate the retardation of CXCR2 internalization in the EGFP-Rab5-S34N–expressing cells (C). Images were processed using Photoshop software. Bars, 10 μm.