Cw0102

A semiquantitative analysis of differentiation was performed on cell-seeded hydrogels via Western blotting as described previously using the following antibodies that were diluted in 3% BSA in TBST buffer: aggrecan (1 : 1000, sc-16492, Santa Cruz Biotechnology, USA), Col II (1 : 5000, ab34712, Abcam, USA), and Sox-9 (1 : 1000, sc-20095, Santa Cruz Biotechnology, USA) antibodies and goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1 : 2000, CW0102, Cwbiotech). And the protein level was quantified and normalized to GAPDH bands by densitometry in Quantity One software (version 4.6.2, Bio-Rad, n = 3).

Cells (washed three times with PBS) and the tissues were lysed in a RIPA lysis buffer (CW2333, CWBIO, China) with protease inhibitor (CW2200, CWBIO, China) and phosphatase inhibitor cocktail (CW2383, CWBIO, China) for 45 min. Protein concentrations of cell lysates and tissue lysates were determined using a Pierce BCA Protein Assay Kit (CW0014, CWBIO, China) according to the manufacturer's instructions. Total protein (30-50 μg) for each sample was separated by the SDS–PAGE and transferred to polyvinylidene difluoride (PVDF, Millipore, IPVH00010) membranes. The membranes were blocked with 5% nonfat milk at room temperature for 2 h and then incubated with primary antibodies overnight at 4°C (for all antibodies, see Supplementary Table2). After washing, the membranes were incubated with a 1 : 8,000 dilution of goat anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (CW0102 and CW0103, CWBIO, China) at room temperature for 1.5 h. Protein bands were visualized using an Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore) via an automatic chemiluminescence imaging analysis system (Tanon-5200, USA). ImageJ (NIH, National Institutes of Health, USA) was used to analyse these images.