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Transfected cells were cultured for 24?hours. Subsequently, cells were serum-starved by culturing in the presence of 2% bovine serum albumin for 4?hours and treated with 2.5% serum from experimental rats for 6?hours. Cells showing either nuclear or cytoplasmic accumulation of ��-catenin were visualized directly http://www.selleck.cn/products/s-gsk1349572.html using a fluorescence microscope. SiRNA ��-catenin (sc-44253) was purchased from Santa Cruz (Santa Cruz BioTechnology, Inc., Santa Cruz, CA, USA), and transfection of siRNA ��-catenin into ST2 cells was carried out using an Amaxa Cell Line Nucleofector Kit (Amaxa Biosystems, Gaithersburg, MD, USA). Right tibial bone tissue proteins for Western immunoblot analysis were extracted using cell lysis buffer, as described previously.18 The cytosolic and nuclear fractions of bone tissues and cells were prepared according to a procedure provided by the manufacturer (Pierce Biotechnology, Rockford, IL, USA). Western blot and immunoprecipitation analyses were performed using standard protocols. Primary and secondary antibodies for ��-catenin, ��-actin, Runx2, Msx2, p38, and ERK1/2 were purchased from Santa Cruz Biotechnology and Cell Signaling (Danvers, MA, USA). Blots were developed http://www.selleckchem.com/products/ly2157299.html using chemiluminescence (Pierce Biotechnology) according to the manufacturer's recommendations. Quantification of the intensity of the bands in the autoradiograms was performed using a VersaDoc imaging system (Bio-Rad). Sera from control or 10% BB diet�Cfed rats were processed with Sep-Pak C18 SPE (Waters, Pittsburgh, PA, USA) cartridge as follows: The cartridge was washed with 3?mL of methanol, followed by equilibration with 3?mL of 0.2% formic http://www.selleckchem.com/products/z-vad-fmk.html acid aqueous solution. Serum (500??L) was loaded onto the cartridge. The cartridge was washed with 3?mL of 0.2% formic acid aqueous solution, and total phenolic acids were recovered with 0.2% formic acid�Cmethanol solution. The methanol solution was dried under N2 flow and redissolved in 200??L of 0.2% formic acid�Cmethanol solution for phenolic acid analysis. Characterization and quantification of phenolic acids were carried out using an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA, USA) coupled with a 4000 Q TRAP mass spectrometer (Applied Biosystems) according to a method described previously.19 Data were expressed as means?��?SEM. ANOVA was used followed by Student-Newman-Keuls post hoc analysis for multiple pairwise comparisons between treatment groups. Values were considered statistically significant at p?