Anti col2a1 antibody

Primary MCs and C28/I2 cells were fixed in 4% PFA for 20 min, permeabilised with 0.1% Triton X‐100 for 30 min, and then blocked with 5% Bovine serum albumin (BSA) at room temperature (RT) for 30 min. The cells were incubated with the following primary antibodies: anti‐LOX1 antibody (1:200, Proteintech, 11837‐1‐AP), anti‐Aggrecan antibody (1:200, Sigma–Aldrich, c8035), anti‐COL2A1 antibody (1:200, Abcam, ab34712), anti‐Vimentin (1:200, Cell Signaling Technology, 5741), and anti‐SYVN1 (1:200, Proteintech, 67488‐1‐Ig). After rinsing with phosphate buffer saline (PBS), the cells were incubated with fluorescent secondary antibodies at RT for 1 h. Subsequently, the cells were rinsed with PBS and stained with DAPI at RT for 20 min. Finally, the target proteins were observed using a fluorescence microscope (NIKON TE2000, Nikon Corporation, Japan).

Isolated chondrocytes were collected for protein extraction using RIPA lysis buffer. A BCA Protein Concentration Assay Kit (Thermo Scientific, USA) was used to determine the protein concentration of the sample according to the manufacturer’s protocol. Proteins were separated on a 10% SDS gel and transferred into PVDF membranes. Primary antibodies were used as follows: anti-KAT6B antibody, 1:1000 dilution, cat no. ab191994, Abcam; anti-RUNX2 antibody, 1: 1000 dilution, cat no. ab76956, Abcam; anti-RUNX3 antibody, 1: 1000 dilution, cat no. ab49117, Abcam; anti-COL2A1 antibody, 1: 100 dilution, cat no. ab239007, Abcam; anti-COL10A1 antibody, 1: 1000 dilution, cat no. ab182563, Abcam; anti-β-catenin antibody, 1:1000 dilution, cat no. ab6302, Abcam; anti-c-Myc antibody, 1:1000 dilution, cat no. ab32072, Abcam; anti-VEGF antibody, 1:1000 dilution, cat no. AB-293-NA, Bio-Techne; anti-GAPDH antibody, 1:3000 dilution, cat no. sc-47724, Santa Cruz Biotechnology. After primary antibody incubation, the membranes were incubated with a secondary anti-rabbit antibody or anti-mouse antibody (1: 5000, Cell Signaling Technology, USA). The bands were measured using a chemiluminescence system (Bio-Rad, USA) imaging system.