Cxcr6

Cells were lysed using RIPA buffer (Biosolution, Korea), phenylmethylsulfonyl fluoride (Sigma), protease inhibitor, and phosphatase inhibitor cocktail 2/3 (Sigma) and then centrifuged at 25,000 x g for 30 min. Cell extracts were quantified using the BCA protein assay kit (Pierce Biotechnology, USA) according to the manufacturer's instructions. The proteins (20-30 μg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (GE Healthcare, UK) for western blot analyses. The transferred membrane was blocked with 1x Tris-buffered saline with Tween 20 (Sigma) (TBST) containing 5% skim milk (BD Biosciences) for 1 h and then incubated with primary antibodies ADAM10 (Santa Cruz), ADAM17 (Abcam, USA), β-arrestin (Bethyl, USA), and CXCR6 (GeneTex) in 1x TBST containing 1% skim milk at 4°C overnight. The membrane was washed three times with 1x TBST for 10 min, then incubated with secondary anti-rabbit (Cell Signaling) and anti-mouse (Santa Cruz) antibodies in 1x TBST containing 1% skim milk for 45 min. The membrane was washed three times with 1x TBST for 15 min and was visualized with enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, USA) and imaged by ChemiDoc (Bio-Rad Laboratories, USA).

Cells were fixed with 4% paraformaldehyde to detect the cell membrane protein and blocked with 1% BSA for 30 min. Cells were then incubated with CXCR6 (GeneTex, USA) or CXCL16 (GeneTex) primary antibodies at 4°C overnight. To detect cells stained by primary antibodies, the cells were incubated with Alexa 488-conjugated anti-rabbit secondary antibodies (Cell Signaling, USA) for 45 min. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Fluorescent images were obtained by confocal microscopy (Nikon) and quantitatively analyzed by the ImageJ program (National Institutes of Health, USA).