Mouse anti col10a1

Mouse anti-COL10A1 (Abcam), rabbit anti-SOX9 (Abcam), anti-E-cadherin, anti-vimentin primary antibodies (CST) and appropriate fluorophore-conjugated secondary antibodies (KeyGEN, Nanjing, China) were used for immunofluorescence labeling. The nucleus was visualized with 1 μg/mL DAPI. Images were then taken using an Olympus confocal microscope (Olympus Fluoview 500 IX71) or a fluorescence microscope.

The proteins were dissociated and separated by SDS/PAGE, and then transferred to PVDF membranes, which were incubated with primary antibodies. The following primary antibodies were used: rabbit anti-SOX9, anti-MMP2, anti-MMP3, anti-MMP9, anti-MMP14, anti-BMP4, anti-TGFβ1, anti-Smad2, anti-Smad3, anti-p-Smad2 (phosphor S255), anti-p-Smad3 (phosphor S423 + S425) and mouse anti-COL10A1 (Abcam, Cambridge, MA, USA); rabbit anti-Beta-catenin, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-Snail, anti-Slug, and anti- GAPDH (Cell Signaling Technology, Danvers, MA, USA). Antigen-antibody complexes were detected using horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) with ECL western blot detection reagent (Merck Millipore). Protein expression was quantified using US National Institutes of Health (NIH) ImageJ software.