Role of Rab11a in CXCR2 recycling.

HEK293 cells stably expressing CXCR2 were transiently transfected with plasmids for vector (A-C) Rab11a (D-F) or Rab11a-S25N (G-I). Cells were treated with CXCL8 at 37°C for 30 minutes, then the agonist was removed and the cells were recovered by incubation with agonist-free medium for 0 minutes (A,D,G), 30 minutes (B,E,H), or 1 hour (C,F,I) at 37°C. For the staining of the cell surface receptor, cells were incubated with a monoclonal CXCR2 antibody at 4°C for 1 hour, followed by incubation with FITC-conjugated antimouse IgG at 4°C for 30 minutes. Cells were washed and fixed in 2% formaldehyde in PBS and analyzed in FACScan. Thin solid line represents the staining of cells in the absence of primary CXCR2 antibody (background). Thick solid line represents the immunostaining of cell surface CXCR2 in the cells without CXCL8 treatment (total). Broken line indicates the immunostaining cell surface CXCR2 in the cells treated with CXCL8 and recovered for different intervals. (J) Relative cell surface fluorescence (percentage of total) of the cells recovered for different intervals was quantified. Data are means ± SEMs of 4 independent experiments (*P < .05).