Anti carbonic anhydrase 2

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).

Soluble recombinant mouse RANKL (sRANKL) was purchased from Peprotech (Rocky Hill, NJ, USA). The primary antibodies used were as follows: anti-TRAP, anti-cathepsin K, anti-c-Fos, anti-NFATc1, anti-Blimp-1, and anti-β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-carbonic anhydrase II, anti-cathepsin K, anti-HDAC6, and anti-IRF-8 from Abcam (Cambridge, MA, USA); and anti-Calcineurin from by MyBiosource (San Diego, CA, USA). Calcineurin inhibitors FK506 and cyclosporin A were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and Abcam (Cambridge, MA, USA), respectively; the inhibitors were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and stored at −20 °C. HDAC6 inhibitor CKD-WID was kindly provided by the Chong Kun Dang Pharmaceutical Corp. (Seoul, Korea).