Phenylmethanesulfonyl fluoride pmsf

Protein was extracted from MC3T3-E1 cells and immunoblotting performed as previously described (Pan et al., 2012 (link)). Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris Base, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% NP-40, pH = 7.4) with 1% protease inhibitor (Calbiochem, CA, USA) and phenylmethane sulfonyl fluoride (PMSF) (Thermo Fisher Scientific, MA, USA) freshly added on the day of the experiment (Calbiochem, Gibbstown, NJ, USA). The harvested protein was incubated on ice for 5 min before centrifuging at 13,000 rpm for 5 min at 4 °C. Total protein concentration was determined with a Nanodrop 2000C Spectrophotometer (Thermo Fisher Scientific, MA, USA) using a bovine serum albumin (BSA) standard curve (Sigma-Aldrich, MO, USA). The expression of FoxO3a, RXRα, VDR and myc was assessed by Western blotting with anti-FoxO3a (D19A7) rabbit mAb (Cell Signaling, MA, USA), anti-retinoid receptor alpha (RXRα) (Abcam, MA, USA), anti-vitamin D receptor (VDR) (Abcam, MA, USA) and anti-c-Myc (Y69) rabbit mAb (Cell Signaling, MA, USA). For internal control, blots were stripped and blotted for β-actin (Santa Cruz Biotechnology, Santa Cruz, CA). Quantification of relative band intensity was performed with Image J Software and Image Lab™ software (Biorad, CA, USA).