Figure 2

K327 residue drives CXCR2 trafficking in response to IL-8. HEK-293T cells were transfected with pCEFL-AU5 plasmid (mock), containing in frame wild type (WT), K327R (R1), and K337R (R2) mutant forms of FL CXCR2. a. CXCR2 staining was analyzed by confocal microscopy in 1 h-starved cells (control) followed by IL-8 stimulation (50 ng/ml, 15 min). b. CXCR2 antibody uptake was performed as described in methods and analyzed by confocal microscopy in control and IL-8 stimulated cells. Internalization was estimated by the percentage of cells with CXCR2 vesicular staining per transfected cells. Results are expressed as the mean ± sem, n = 150 cells. c. Alternatively, IL-8-challenged cells were co-transfected with either Rab7-GFP or AP2-GFP 24 h prior confocal analysis. Pearson’s correlation coefficient (P. coef) was calculated on 5 and 3 samples for Rab7-GFP and AP2-GFP, respectively. Each panel is representative of three independent experiments. Scale bars: 10 μm. **p < 0.01, ***p < 0.001.