Runx2 antibody

The caput femoris samples were milled and lysed with RIPA lysis buffer (Beyotime, China) for extracting total proteins. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked for 2 h at room temperature in TBS-Tween 20 (TBST) buffer containing 5% BSA, washed with TBST three times, and incubated overnight at 4°C with 1/500 dilution of Wnt10b antibodies, LRP5 antibodies, β-catenin antibodies, Axin2 antibodies, OPG antibodies, RANKL antibodies, Dkk-1 antibodies, SOST antibodies, Runx2 antibodies, PPAR-γ antibodies, C/EBPα antibodies, FABP4 antibodies (all purchased from Santa Cruz Biotech), and GAPDH antibody (1 : 1000, BOSTER, China), respectively. After being washed with TBST, the membranes were incubated with the secondary biotin-conjugated antibody and then with anti-biotin horseradish peroxidase- (HRP-) linked antibody (1 : 1000). Protein signals were detected using SuperSignal West Pico Chemiluminescent Substrate Trial kit (Thermo Scientific) and quantified by densitometry using Quantity One software (Bio-Rad).

Anti-Cx43 and Runx2 antibodies were obtained from Santa Cruz Biotechnology. Anti-ERK1/2, phospho-ERK1/2, Smad2/3, phospho-Smad2/3(465/467), SAPK, phospho-SAPK, p38, phospho-p38, and HPRP-conjugated anti-rabbit IgG were obtained from Cell Signaling Technology. Anti-phospho-serine antibodies were purchased from Sigma-Aldrich. The anti-AMBN antibody has been previously described (Fukumoto et al., 2004 (link)). Alexa-488 or 594 conjugated anti-rabbit IgG was purchased from Molecular Probes. 18α-glycyrrhetinic acid (18α-GA), oleamide, adenophostin-A, and 2-APB were obtained from Sigma-Aldrich. PD98059 was obtained from Cell Signaling Technology. Fura-2AM was obtained from Invitrogen. TGF-β1, BMP2, and BMP4 were obtained from R&D Systems. Briefly, the pEF6/Cx43 vector was constructed by cloning the coding sequence of mouse Cx43 cDNA into the pEF6/V5-His TOPO vector (Invitrogen). Cx43 expression vectors carrying R76S or R202H mutations were prepared using a Quick Change Site-Directed Mutagenesis Kit (Stratagene). siRNA for Cx43 was purchased from Invitrogen.