Any kdtm mini protean tgxtm gels

Both MDA-MB-231T and MDA-MB-435 cells were twice washed in 1X Phosphate-buffered saline. Cells were (10 cm² dish) lysed in 100 μl RIPA buffer (20mM Tris-HCl, pH 8.0; 100mM NaCl; 10% Glycerol; 1% NP-40; 0.5% Sodium deoxycholate; 0.1% SDS; Protease Inhibitor Cocktail) added directly on cells and were incubated on ice for 10 min. Cells were scraped and clarified by spinning at 10,000 rpm for 10 min at 4°C. supernatants were collected and protein estimation was performed by BCA analysis (Pierce^® BCA Protein Assay Reagent A and B- Prod# 23228). Lysates were frozen at −80°C until use. Equal amount of protein was loaded on Any KD^TM Mini-PROTEAN TGX^TM Gels (BIO-RAD, Cat# 456-9033).

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.