Agonist-induced colocalization of CXCR2 with transferrin and LDL in HEK293 cells.

Cells stably expressing CXCR2 were incubated with CXCL8 (200 nM) and transferrin–Texas Red (25 μg/mL) (A-B) or Dil-LDL (40 μg/mL) (C, D) at 37°C for 1 hour (A, C) or 4 hours (B, D). Cells were washed in cold PBS and fixed in methanol. Cells were incubated with a monoclonal CXCR2 antibody for 30 minutes at room temperature, followed by incubation with an FITC-conjugated antimouse antibody for 30 minutes at room temperature. Representative laser-scanning confocal micrographs from 3 independent experiments demonstrating the distribution of CXCR2 (green), transferrin–Texas Red (red), Dil-LDL (red), and colocalization of CXCR2 with transferrin–Texas Red (yellow; A-B) or CXCR2 with Dil-LDL (yellow; C-D) are shown. Bars, 10 μm. (E) Quantification of the percentage of CXCR2 colocalized with transferrin (■) or LDL (▪) was determined by counting the colocalized receptor fluorescence (yellow) from the total internalized receptor fluorescence (green and yellow). Data are means ± SEMs of 3 independent experiments (**P < .01). Tf indicates transferrin.