Dura luminol substrate

Cells were harvested by scraping the cells from cultured dishes using a cell scraper. Cellular lysates were prepared using RIPA cell lysis buffer. The cells were disrupted and extracted at 4°C for 30 minutes. After centrifugation at 16,000 ×g for 15 minutes, the supernatant was obtained as the cell lysate. Protein concentrations were measured using a protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Cellular proteins (30 μg) were subjected to 10% SDS-PAGE. The resolved proteins were transferred to an Immobilon-P-membrane and allowed to react with a specific antibody. The detection of specific proteins was carried out by Super-signal pico-chemiluminescent substrate or dura-luminol substrate (Thermo Scientific, Waltham, MA, USA) according to manufacturer’s instruction and visualized with imagequant LAS 4000 (Fujifilm Life Science, Tokyo, Japan). Loading differences were normalized using anti-β-actin antibody.

For detection of CXCR4, OBW-treated whole cell extracts were lysed with RIPA buffer (150 mM NaCl, 10 mM Tris [pH 7.2], 0.1% sodium dodecyl sulfate [SDS], 1% triton X-100, 1% deoxycholate, and 5 mM EDTA) enriched with a complete protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany), and then incubated on ice for 30 minutes with regular vortex before centrifuging at 14 000 rpm at 4°C for 15 minutes. Protein concentration was determined by using bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL). The protein samples were boiled in SDS sample buffer for 5 minutes and were resolved on a 10% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred onto polyvinyl difluoride (PVDF) membrane, which was blocked with 5% nonfat dry milk in tris-buffered saline with 0.1% tween-20 (TBST) and incubated with primary antibody at the appropriate final concentration followed by hybridization with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies. For each step, the membrane was washed with TBST 3 times for 10 minutes and the transferred proteins were incubated with super-signal pico-chemiluminescent substrate or dura-luminol substrate (Thermo Scientific, Waltham, MA) for 2 minutes according to the manufacturer’s instruction and visualized with imagequant LAS 4000 (Fujifilm Life Science, Roche Diagnostics).