Figure 4

K327R mutation abrogates CXCR2 signaling 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. One day later, cells were starved (control) or exposed to IL-8 (50 ng/ml, 15 min, unless specified). a. Anti-phospho-Tyr (4G10), anti-CXCR2 western-blots were performed in CXCR2 immunoprecipitated (IP) fraction. b. Calcium flux was measured using the Fluo4NW probe in response to IL-8 for the indicated times. Fluorescent units were normalized to control conditions. c. Anti-AU5, anti-pERK1/2 and anti-ERK2 western-blots were performed. Pixel intensities of the pERK1/2 lanes were quantified using Image J software and normalized to total ERK2. d. Promoter activity of both NF-κB and AP1 was monitored through luciferase-based assays. Each panel is representative of three independent experiments. Means + sem are shown. **p < 0.01, ***p < 0.001.