Protease inhibitor cocktail 1 tablet

Total proteins were obtained by using radio immunoprecipitation assay (RIPA) buffer (Thermo Scientific, Waltham, MA, USA) containing a protease inhibitor cocktail (protease inhibitor cocktail 1 tablet; Roche Diagnostics, Mannheim, Germany) and centrifuged at 13,000 rpm for 10 min. The protein was measured with BCA Protein Assay Kit by using microplate reader at 562 nm. Equal amounts of protein were separated on 10% SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% bovine serum albumin, protein was probed with appropriate antibodies.
The primary antibodies used were as follows: anti-TRAP, anti-cathepsin K, anti-c-Fos, anti-NFATc1, anti-TRAF6, and anti-β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-carbonic anhydrase II, anti-IκBα, and anti-phospho-IκBα from Abcam (Cambridge, MA, USA); and anti-IL-1β and anti-cleaved IL-1β from Cell Signaling Technology (Danvers, MA, USA). The SuperSignal® West Pico chemiluminescent kit (Thermo Scientific, Rockford, IL, USA) was used to detect the target protein. Images were obtained using a ChemiDoc TM XRS system (Bio-Rad Laboratories, Hercules, CA, USA).

Cells (2 × 10⁵) were seeded in 60 mm dishes and cultured with sRANKL (100 ng/mL) and MSU crystals (0.1 mg/mL) for 4 days, followed by treatment with cyclosporin A, FK506, or CKD-WID for 4 days. Cells were lysed using radio immunoprecipitation assay (RIPA) buffer (Pierce, Thermo Scientific, Waltham, MA, USA) containing a protease inhibitor cocktail 1 tablet (Roche Diagnostics, Mannheim, Germany) on ice for 10 min and centrifuged at 13,000 rpm for 10 min. The supernatant was collected and stored at −80 °C. Cytosolic and nuclear fractions were separated using the NE-PER™ nuclear and cytoplasmic extraction reagents (Thermo Scientific, Rockford, IL, USA) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) in accordance with the manufacturer’s instructions. The samples were mixed with 6× Laemmli Buffer (Bio-Rad, Hercules, CA, USA) and denatured by boiling at 95 °C for 5 min, followed by separation by 10% SDS-PAGE gel electrophoresis and transfer to a nitrocellulose membrane (Bio-Rad). The membrane was probed with appropriate antibodies.