4 to 12 gradient bis tris gel

Media and cell lysate from each time point (d3, d7, d11, d14, d18, d21) were homogenized in lysis buffer (10 mmol/L Tris base, pH 7.4; 150 mmol/L NaCl; 1 mmol/L EDTA; 1 mmol/L EGTA; 0.1% Triton X-100; 0.5% Nonidet P-40; and protease and phosphatase inhibitors) and sonicated. Total protein concentrations were determined using a Bradford assay kit (Bio-Rad, Hercules, CA), ran in 4 to 12% gradient Bis-Tris gels (Life Technologies, Carlsbad, CA) and then transferred to polyvinylidene difluoride (PVDF) membrane. Once transferred, membranes were processed according to methods previously published (Ramser et al., 2021). Primary antibodies used were rabbit anti-COLI (Origene, Rockville, MD), mouse anti-COLII (Novis Biologicals, Littleton, CO), rabbit anti-ACAN (ABClonal, Woburn, MA), rabbit anti-Sox9 (ABClonal, Woburn, MA), and rabbit anti-COL10A1 (ABClonal, Woburn, MA). The protein signals were visualized using chemiluminescence (ECL Plus; GE Healthcare, Pittsburg, PA) and images were captured using the FluorChem M MultiFluor System (ProteinSimple, San Jose, CA). Protein loading was assessed by immunoblotting using rabbit antieglyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, Dallas, TX) or Ponceau S Stain (G-Biosciences, St. Louis, MO). Image acquisition and analysis were performed by AlphaView software (version 3.4.0, 1993–2011; ProteinSimple, San Jose, CA).