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4 protocols using anti col2a1 antibody

1

Characterization of Chondrocyte Markers

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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).
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2

Chondrocyte Protein Expression Analysis

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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.
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3

Immunohistochemical Analysis of COL2A1

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Paraformaldehyde (4%) was used for fixation of the tissues at room temperature for 1 h. Paraffin sections (4-µm thick) were prepared and 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used as the blocking reagent for 20 min at room temperature. IHC was performed using the Histostain-Plus kit (Origin Technologies, Inc., Beijing, China) with the anti-COL2A1 antibody (Abcam, 1:500) at 4°C overnight. Detection was conducted using a DAB Horseradish Peroxidase Color Development kit (Origin Technologies, Inc.). Semi-quantitative analysis of IHC images through integrated optical density (IOD) was performed using Image-Pro Plus 7 software (Media Cybernetics, Inc., Rockville, MD, USA).
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4

Protein Expression Analysis in Cartilage Tissue

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The cells were washed with cold PBS 3 times; total protein was extracted with RIPA buffer containing 1% PMSF, and the protein concentration was measured with a quantitative BCA protein kit (Thermo Fisher, USA). The following primary antibodies were used:anti-COL 2A1 antibody (1:1000, Abcam); anti-ACAN antibody (1:1000, Abcam); anti-BMP-2 antibody (1:1000, Abcam); anti-ITG A1 antibody (1:1000, Abcam), anti-Smad1 antibody (1:1000, Abcam), anti-RUNX2 antibody (1:1000, Abcam), and anti-GAPDH antibody (1:1000, Abcam). GAPDH was used for normalization of the data. The membrane was incubated with the corresponding horseradish peroxidase (HRP)-labeled secondary immunoglobulin G conjugate (1:2000, Abcam) at room temperature for 1 h. Protein bands were visualized and detected using an enhanced chemiluminescence (Thermo Fisher, USA) system. The experiments were performed in triplicate.
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