Bmp 2

At each indicated time point, cells from each group were washed twice with ice-cold PBS supplemented with 1 mM sodium vanadate and lysed in modified radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1 mM EGTA, 50 mM Tris (pH 7.4), 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail (Complete Protease Inhibitor Cocktail Tablets; Roche Diagnostics Ltd., Taipei, Taiwan) and 1 mM sodium vanadate. The lysates were cleared by centrifugation at 14,000 rpm for 15 min at 4 °C. The proteins were quantitated using the BCA protein assay. The protein expression levels were analyzed by Western blotting using antibodies against BMP-2 (catalog number: bs-1012R; Bioss, Beijing, China), anti-p38 (catalog number: 14064-1-AP; Proteintech, Rosemont, IL, USA), anti-p-p38 (catalog number: AP0526; ABclonal, Wuhan, China), anti-JNK (catalog number: ARG51218; Arigo, Hsinchu, Taiwan), anti-p-JNK (catalog number: ARG51807; Arigo, Hsinchu, Taiwan) and β-actin (catalog number: A5441), and immunoreactions were visualized using an enhanced chemiluminescence (ECL) system (Amersham, UK).

Proteins were extracted from cells by total proteins extraction kit (Tiangen, China) according the manuscripture. Total proteins were separated on a 12% SDS-PAGE, and transferred to a polyvinylidene fluoride membrane (Beyotime, China). The membrane was blocked in 5% BSA blocking solution for 1 h at room temperature and incubated overnight at 4°C with primary antibodies (Bioss, China) as follows: BMP6 (bs-10090R, diluted 1:500), Collagen II (bs-10589R, diluted 1:500), Collagen X (bs-0554R, diluted 1:500), BMP2 (bs-10696R, diluted 1:500), JAK2 (bs-0908R, diluted 1:500), PKC (bs-3729R, diluted 1:500), IHH (bs-6624R, diluted 1:500), PTHrP (bs-1107R, diluted 1:500), IGF1R (bs-0680R, diluted 1:500) and beta-actin (bsm-33036M, diluted 1:1,000). After that, the membrane was washed with scrubbing solution (Beyotime, China) and then developed with anti-mouse or rabbit horseradish peroxidase conjugated secondary antibodies (Beyotime, China, diluted 1:1,000). Protein bands were visualized using enhanced chemiluminescence (ECL) system (Beyotime, China) and quantified with an Image Lab system (Bio-Rad, USA).

CS was supplied by Zhengzhou Corey Fine Chemical (MW 30–36 kDa) (with 55% deacetylation), TGF-β3 and RHC were provided by Jinan University Biopharmaceutical R and D Center (Guangzhou, China), and α-modified minimum essential medium (α-MEM), fetal bovine serum (FBS), and trypsin-EDTA were purchased from Gibco BRL. Penicillin and streptomycin (P/S) were purchased from MD Bio, China. MTT was purchased from MP Biomedicals (United States). Antibodies against collagen-1 (COL Ⅰ) were purchased from Affinity Biosciences (Cincinnati, OH, United States), BMP-2 and RUNX2 were purchased from Bioss (Boston, MA, United States), and GAPDH and an horseradish peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology (Boston, MA, United States).