Figure 1

K327 residue is critical for IL8-induced CXCR2 ubiquitination. a. Partial sequence alignment between Homo sapiens (h), Mus musculus, Gallus gallus, and Sus scrofa CXCR2 (ILRB), and between hCXCR2 and hCXCR1 (ILRA). Conserved residues are in black, not conserved one are in grey. Conserved lysines are highlighted in red. b. CXCR2 secondary structure was predicted using LOMETS software and modeled using Swiss-PDB viewer. K327 and K337 are indicated with an arrow and colored in red. c-g. HEK-293T cells were transfected with pCEFL-AU5 plasmid (mock), in frame wild type (WT), K327R (R1), and K337R (R2) mutant forms of FL CXCR2. c. Anti-AU5 and anti-Tubulin western-blots were performed 24 h later. d. CXCR2 immunoprecipitation (IP) was performed onto SDS denaturated protein lysates from IL-8-treated samples (15 min, 50 ng/ml), and further analyzed by western-blots for CXCR2, β-arrestin and CXCR2. Input for CXCR2 is also presented. e-f. Alternatively, cells were pre-incubated with CXCR2 antagonist (SB225002, 50 μm, 1 hour) or MG132 proteasome inhibitor (25 μm, 45 min). g. CXCR2 IP and WB were performed as in (d) in starved cells (-) and in IL-8-challenged cells (+, 15 min, 50 ng/ml). Pixel intensities of the ubiquitination lanes were quantified using Image J software and normalized to untreated mock lane. Means + sem are shown. Each panel is representative of three independent experiments. *p < 0.05.