Recycling of CXCR2 in HEK293 cells overexpressing EGFP-Rab11a-S20V or EGFP-Rab11a-S25N.

Cells coexpressing CXCR2 and Rab11a-S20V (A-D) or Rab11a-S25N (E-H) were treated with CXCL8 at 37°C for 30 minutes, then the agonist was removed. Cells either were fixed immediately (A-B,E-F) or were incubated with agonist-free medium for 1 hour at 37°C before being fixed (C-D,G-H). 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 4 independent experiments demonstrating the intracellular distribution of CXCR2 (A,C,E,G) and EGFP-Rab11a-S20V (B,D) or EGFP-Rab11a-S25N (F,H) were shown. Small arrows indicate the recycled CXCR2 on the cell surface in the cells expressing EGFP-Rab11a-S20V (C). Large arrow indicates the nonrecycling of CXCR2 in the EGFP-Rab11a-S25N-expressing cell (G). Images were processed using Photoshop software. Bars, 10 μm.