Mouse anti nfatc1

0.4-μM Transwells were bought from Costar Corp. (USA). Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), lactic dehydrogenase (LDH) in patients were determined by routine laboratory tests in Anhui Provincial Hospital (Hefei, China). Kits for serum Hydroxyproline (Hyp) was bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Recombinant Human Wnt 5a was bought from R&D Systems (645-WN-010/CF). Viability of Hepatic stellate cell line (LX-2) cells was detected by Cyto Tox 96 Nonradioactive Cytotoxicity assay from Promega (USA). The primary antibodies were as following: rabbit anti-Jnk (#9252, Cell signaling), rabbit anti-pJnk (Thr 183/Tyr 185, #4668, Cell signaling), mouse anti-α-SMA (ab5694, Abcam), mouse anti-NFATc1 (sc-7294, Santa Cruz), rabbit anti-Collagen 1A1 (MA1-141, ThermoFisher Scientific, USA), mouse anti-β-actin (ab8227, Abcam). Horseradish peroxidase conjugated secondary antibodies were obtained from GE Healthcare (UK).

The protein from homogenized bone tissue from hamster were isolated using Trizol reagent (Thermo Fisher, USA) following the manufacturer’s instructions. The protein from cultured BMM was harvested using RIPA Lysis and Extraction Buffer (Thermo Fisher, USA) containing a 1% Phosphatase Inhibitor Cocktail (Thermo Fisher, USA). The concentration of protein was measured using the BCA Protein Assay Kit (Thermo Fisher, USA). A total of 20 µg of protein from each sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the polyvinylidene difluoride (PVDF) membrane (Merck Millipore, USA). Then, the membrane was blocked in 5% w/v bovine serum albumin (BSA, Sigma-Aldrich, USA) and incubated with blocking buffer-diluted primary antibodies overnight at 4 °C. The primary antibodies used include mouse anti-NFATc1 (Santa Cruz, USA), mouse anti-TRAP (Abcam), rabbit anti-Cathepsin K (Abcam), mouse anti-RANK (Abcam), rabbit anti-NF-κB p65 (CST), rabbit anti-IL-1β (Abcam), rabbit anti-IL-1RA (Abcam), rabbit anti-TNF-α (Abcam), rabbit anti-phospho-JNK (CST), rabbit anti-JNK (CST), rabbit anti-β-actin (Abcam). The protein bands were visualized by using HRP conjugated secondary antibodies and an enhanced chemiluminescence (ECL) substrate (Advansta, USA) and exposed under the Typhoon5 Biomolecular Imager 680 (GE Amersham, USA).

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM tris-HCl pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, and 1 mg/ml leupeptin). Protein concentrations were determined using the bicinchoninic acid (BCA) assay kit (Pierce). Protein was resolved by 10-12% sodium SDS-PAGE and transferred to Hybond-ECL nitrocellulose membranes (Amersham Biosciences). Membranes were blocked with 5% skim milk in tris-buffered saline at room temperature for 1 hour, and then probed with indicated antibodies (1:1000) at 4°C overnight and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. Immunoblots of protein bands were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham). The used antibodies were as follows: Rabbit anti-Sema3A, Rabbit anti- pSTAT3 (Y705), Mouse anti-STAT3, Mouse anti-β-actin (Abcam), Mouse anti-NFATc1 (Santa cruz), Rabbit anti-c-Src, Rabbit anti-integrin β3 (Cell Signaling), Rabbit anti-cathepsin K (Invitrogen).