Af5166

Four weeks after surgery, three rats from each group were sacrificed, and subchondral bone was analyzed by western blotting. Articular cartilage was removed and subchondral bone from the proximal tibia was preserved. The tissues were lysed using a buffer containing protease inhibitors and phosphatase inhibitors and pulverized in an automated frozen sample grinder (Jingxin Industrial Development Co., Ltd; Shanghai, China). Protein concentrations were measured using a kit (KGP250; Keygen Biotech Co., Ltd., Nanjing, China), proteins were resolved using 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then electroblotted onto polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). Non-specific protein binding was blocked using skim milk (Fujifilm, Tokyo, Japan) for 2 h, and then the membranes were incubated overnight with primary antibodies against Wnt5a (1:1000, 55184-1-AP), RANKL (1:1000, 23408-1-AP; both from Proteintech), CXCL12 (1:1000, AF5166; Affinity Biosciences, Cincinnati, OH, USA), NFATc1 (1:5000, ab264530; Abcam), and β-actin (1:5000, 20536-1-AP; Proteintech), followed by goat anti-rabbit Ig–G conjugated HRP (1:10000, SA00001-2; Proteintech). Signals were detected using enhanced chemiluminescence. Data are shown relative to the intensity of the β-actin control.

The proteins from the callus tissues were extracted by using lysis buffer (Beyotime, Shanghai, China). Then the protein was quantified by the BCA assay kit (Beyotime, Shanghai, China). After that, an equal amount of proteins was electrophoresed on SDS-PAGE and then transferred onto PVDF membranes (Thermo Fisher, Cambridge, MA, USA). Next, the membranes were immuno-blotted with Hypoxia-inducible factor-1α (HIF-1α) rabbit antibody (1: 500 dilution, Catalog No.: AF1009, Affinity, Changzhou, Jiangsu, China), vascular endothelial growth factor (VEGF) rabbit antibody (1: 500 dilution, Catalog No.: bs-1313R, Bioss, Beijing, China), Runx2 rabbit antibody (1: 500 dilution, Catalog No.: AF5186, Affinity), Osterix (1: 400 dilution, Catalog No.: DF7731, Affinity), Type I collagen antibody (1: 1000 dilution, Catalog No.: AF7001, Affinity), SDF-1α rabbit antibody (1: 500 dilution, Catalog No.: AF5166, Affinity), CXCR4 antibody (1: 1000 dilution, Catalog No.: A12534, ABclonal, Wuhan, China), or β-actin mouse antibody (1: 2000 dilution, Catalog No.:60008-1-Ig, proteintech, Wuhan, Hubei, China). The membranes were washed three times and incubated with goat HRP-conjugated secondary antibodies (proteintech, Wuhan, Hubei, China). The specific bands were developed with the ECL system (7 Sea biotech, Shanghai, China). β-actin was used as the control of HIF-1α and VEGF.