Irdye goat anti rabbit igg

Expression of membrane-bound RANKL was detected by western blot analysis. Chondrocytes were resuspended in Mammalian Protein Extraction Buffer (GE Healthcare, Piscataway, NJ, USA) containing protease inhibitors (diluted 1:100, Takara Bio USA, CA, USA) 60 hours after cytokine stimulation. Protein concentrations were determined by the Bradford method. Similar amounts of protein extracts were loaded onto sodium dodecyl sulfate–polyacrylamide gels (Any kD^TM Mini-PROTEAN^® TGX^TM Gels (Bio-Rad, Munchen, Germany)) and run for 45 minutes at 150 V before transfer to polyvinylidene difluoride membranes using a Trans-Blot^® Turbo^TM Blotting System (Bio-Rad). The membranes were incubated with blocking reagent (Toyobo) and incubated overnight at 4°C with RANKL antibodies (dilution, 1:500; cat. no. ab9957; Abcam). After washing with washing buffer, the membranes were incubated with IRDye Goat anti-Rabbit IgG (LI-COR Biosciences, Lincoln, NE, USA) secondary antibodies at room temperature for 1 hour. Immunoreactive proteins were detected using the OdysseyFc Imaging System (LI-COR Biosciences). We analyzed the densities of the resulting protein bands using the OdysseyFc Imaging System. The levels of membrane-bound RANKL were expressed as ratios and normalized to β-actin.