Cxcr4

RIPA buffer containing proteinase inhibitors (Beyotime, China) was used to create tissue extracts, which were then used to perform the analyses., and the concentration of protein was measured by BCA Protein Assay Reagent (Beyotime, China). The tissue lysate containing 40μg of total protein was loaded onto the SDS-PAGE gel and electrophoresed at 120V for 90 minutes, and then the protein to be tested in the gel was transferred to a 0.45m polyvinylidene fluoride membrane (Merck Millipore, Germany). After blocking the membrane in TBST buffer containing 5% skimmed milk powder for 30 minutes, the membranes were incubated for 12 hours at 4 °C with the following antibodies: CXCR4 (Abcam, UK), GAPDH (Proteintech, China), β-tubulin (Proteintech, China), ERK1/2 (Abcam, UK), p-ERK1/2 (Abcam, UK). The membranes were rinsed three times with TBST (5 minutes/wash) the next day before being incubated with goat anti-rabbit secondary antibody (Thermo Fisher, USA) or goat anti-mouse secondary antibody (Thermo Fisher, USA) in 5% skimmed milk powder /TBST, and then rinsed 3 times. Membrane development was carried out using ECL Substrate (Thermo Fisher, USA), and the chemiluminescence was captured using the bioanalytical imaging system C300 (Azure Biosystems, USA) and analyzed using ImageJ software.

Protein samples were separated by 8% or 10% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). After blocking with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBS-T) for 2 h at 37 °C, the membranes were incubated with primary antibodies for CXCR4 (1:100 dilution), Sox-9 (1:1000 dilution; Abcam), aggrecan (1:1000 dilution), collagen II (1:1000 dilution), and GAPDH (1:10000 dilution; Abcam) overnight at 4 °C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam) for 1 h at room temperature. An electrochemiluminescence reagent was added to the surfaces of membranes, and immunolabeling was visualized using an ImageQuant LAS4000 (GE Healthcare, United States). The relative protein concentrations were measured using Image-Pro Plus 6.0 software. The expression level of GAPDH was used as an internal control.