Mouse anti col1

The expression levels of SOX9, COL2, and COL1 proteins from cell samples were analyzed as described previously [38 (link)]. Samples were lysed in RIPA lysis buffer at 4°C. Samples with equal protein concentration were subjected to SDS-PAGE and transferred to a PVDF membrane. Blots were blocked with 5% skim milk/TBS-Tween 20 for 1 h at room temperature and probed with primary antibodies: mouse anti-SOX9 (Santa Cruz Biotechnology, CA) and mouse anti-COL1 (Abcam, MA) and mouse anti-COL2 (Abcam, MA) and mouse anti-β actin (Abcam, MA) overnight at 4°C. Followed by which blots were washed with PBS-Tween 20 (0.1%) and incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 1000) for 1 h at room temperature. Western blotting images are representative of N ≥ 3 images.

The expression levels of SOX9, COL2, and type I collagen (COL1) proteins from cell samples were analyzed as described previously [39 (link)]. Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, and 0.1% sodium dodecyl sulfate) supplemented with protease inhibitor cocktail set I (Biotool, Jupiter, FL, USA) and phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich, USA). Samples with equal protein concentration (about 80 μg/lane) were subjected to SDS-PAGE and transferred to a PVDF membrane. Blots were blocked with 5% skim milk/TBS-Tween 20 for 1 hr at room temperature and probed with primary antibodies: mouse anti-SOX9 (Santa Cruz Biotechnology, USA), mouse anti-COL1 (Abcam, UK), mouse anti-COL2 (Abcam, UK), and mouse anti-β-actin (Abcam, UK) overnight at 4°C. Then, blots were washed with PBS-Tween 20 (0.1%) and incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 1000) for 1 hr at room temperature.