Ab6302

CFs were washed with PBS (0.1 mol/L, pH 7-7.4) three times, lysed in a lysis buffer for 30 min at 4 °C, and then scraped off using a cell scraper before being mixed with a one-quarter volume of 5× loading buffer. The cell protein mixture was then boiled in a 100 °C water bath and cooled to room temperature. The protein concentration was determined using a Bradford protein assay kit (Amresco, M173-KIT). Equal amounts of protein (50 µg) were separated on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The strips were blocked with 5% nonfat milk for 1 h at room temperature. The strips were then incubated overnight with primary antibodies against AAT1 (Sigma, AV48205, 1:500), collagen I (Abcam, ab254113, 1:500), collagen III (Abcam, ab7778, 1:500), α-SMA (Abcam, ab5694, 1:500), β-catenin (Abcam, ab6302, 1:500), p-P38 MAPK (Abcam, ab4822, 1:500), P38 MAPK (Abcam, ab170099, 1:500) and GAPDH (Abcam, ab181602, 1:2000) diluted in PBS with 0.05% Tween 20 at 4 °C. The strips were rinsed using PBS with 0.05% Tween 20 for 30 min, followed by incubation with secondary antibodies for 1 h at room temperature. All protein bands were detected using Amersham ECL Western blotting detection reagents (GE Healthcare, RPN2106) and analyzed with a biological electrophoresis image analysis system.

SaOS-2 cell was kept in the laboratory. GKC (20180521) was obtained from Guizhou Wei Kang Zi Fan Pharmaceutical Co. Ltd. (Guiyang, China). Jiegu-Qili tablet was obtained from Hunan Jin Sha Pharmaceutical Co. Ltd. (Changsha, China). Pentobarbital Sodium (6900183) was purchased from Beijing Solarbio Science & Technology Co. Ltd. Penicillin G Sodium (170907) was from Lukang Pharmaceutical Co. Ltd. (Jining, China). ALP (A059-2), Pi (C006-3), and Ca (C004-2) were supplied by Nanjing Jiancheng Biotechnology Co. Ltd. (Nanjing, China). Primers of ALP, COL-I, OTC, Osterix, RUNX2, BMP2, OPN, OPG, RANKL, and GAPDH were obtained from Shanghai Generay Biotech Co. Ltd. (Shanghai, China). Antibodies against RUNX-2 (ab23981), OPG (ab73400), BMP2 (ab14933), RANKL (ab9957), β-catenin (ab6302), Smad4 (ab40759), GAPDH (ab8245), and GSK3β (ab93926) were from Abcam (Cambridge, England). DKK1 (PHC9214), Noggin (PHC1506), antibodies against Smad1/5 (PA5-80036), and p-Smad1/5 (MA5-15124) were obtained from Thermo fisher (American). p-GSK3β (Ser9, 9336S) antibody was got from Cell Signaling Technology (American). All other reagents used in the present study were of analytical grade.

Cells were seeded in 75 cm² bottles and after 24 h incubation to attach and stretch, treated with the different compounds. After 40 h cells were washed with cold HANK’s balanced salt solution and lysed and scraped with RIPA buffer [5 (link)]. After 20 min incubation on ice, samples were centrifuged for 15 min at 16,000 g and 4 °C. Protein concentration was determined using Bio-Rad Dc Protein Assay (Bio-Rad Laboratories). Twenty micrograms of protein from total cell lysates was subjected to SDS-PAGE and analyzed by Western blot. Primary antibodies used in this study were directed against β-Catenin (Ab6302 1:4000) (Abcam, Cambridge, UK), HER2 (PA5-14635 1:500) (Pierce-Thermo Scientific), HER3 (PA1-86644 1:2500 (Thermo Scientific), with β-Actin pan Ab-5 (MS-1295-P1 1:2000) (Thermo Scientific) as a reference protein. And as secondary antibody, goat anti-mouse HRP-conjugated (HAF007), goat anti-rabbit HRP conjugated (HAF008) and donkey anti-goat HRP conjugated (HAF109) (R&D Systems, Abingdon, UK) was used in a 1:20.000 dilution. HRP was visualized using Advance TM_Enhanced chemiluminescence (ECL, Amersham, GE Healthcare, Eindhoven, The Netherlands) and analyzed using GelDoc 2000 (Bio Rad). With the Quantity One software, version 4.6.9 (BioRad), densities were measured, corrected for the background and related to β-Actin expression as loading control.