Cells were scraped and lysed on days 3, 5 and 7 during differentiation in lysis buffer at pH 7.4, consisting of 10 mM Trisma-base, 300 mM NaCl, 2 mM EDTA and 0.5% Triton-X-100 with 1 mini protease inhibitor tablet. Samples were then sonicated 10 times at 50% amplitude for 20 s and centrifuged at 17000 g for 3 min at 4 °C. Protein concentration was determined using the bicinchoninic acid assay.
Proteins were denatured with 2× Laemmli buffer and boiled at 100 °C for 5 min. The samples were run on 10% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel and blotted onto nitrocellulose membrane (ThermoFisher Scientific). The membranes were blocked with 10% non-fat dry milk in Tris-buffered saline with 0.2% Tween-20 (0.2% TBS-T) for 1 h. The following antibodies were used: anti-HAPLN1 (1:1000; Novus Biologicals NBP1–59150), rabbit polyclonal anti-ACAN (1:500; Merck AB1031), mouse monoclonal anti-TNR (clone #619 MAB1624, 1:500; R&D Systems), and mouse monoclonal anti-GAPDH (1:5000; Sigma). The blots were incubated in primary antibody overnight at 4 °C, and then incubated with horseradish peroxidase-coupled secondary antibodies (Invitrogen) at a dilution of 1:1000 for 1 h at room temperature (RT; 23 °C). They were then rinsed three times in 0.2% TBS-T, and the signals were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific).
Proteins were denatured with 2× Laemmli buffer and boiled at 100 °C for 5 min. The samples were run on 10% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel and blotted onto nitrocellulose membrane (ThermoFisher Scientific). The membranes were blocked with 10% non-fat dry milk in Tris-buffered saline with 0.2% Tween-20 (0.2% TBS-T) for 1 h. The following antibodies were used: anti-HAPLN1 (1:1000; Novus Biologicals NBP1–59150), rabbit polyclonal anti-ACAN (1:500; Merck AB1031), mouse monoclonal anti-TNR (clone #619 MAB1624, 1:500; R&D Systems), and mouse monoclonal anti-GAPDH (1:5000; Sigma). The blots were incubated in primary antibody overnight at 4 °C, and then incubated with horseradish peroxidase-coupled secondary antibodies (Invitrogen) at a dilution of 1:1000 for 1 h at room temperature (RT; 23 °C). They were then rinsed three times in 0.2% TBS-T, and the signals were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific).