Bca protein quantitative analysis kit

For total protein extraction, we added 60–100 μL of Radio‐Immunoprecipitation Assay (RIPA) buffer (Yeasen Biotech, Shanghai, China) containing 1% protease inhibitor (Yeasen) onto cells seeded in 6‐well plates and incubated on ice for 30 minutes. The cells were scraped, and the cell‐RIPA mixture was centrifuged at ~13 400 g for 10 min at 4°C to rid of cell debris. Protein concentration was determined using bicinchoninic acid (BCA) protein quantitative analysis kit (Beyotime Biotech, Shanghai, China). Protein samples were loaded on a 10% SDS‐PAGE and transferred to 0.22 μm polyvinyl difluoride (PVDF) membranes (EMD Millipore, Merck, Darmstadt, Germany). The membranes were then incubated with the primary antibodies at 4°C overnight. The information on primary antibodies is listed in Table S3. After the membranes were incubated with secondary antibodies on a shaker for 1.5 h at room temperature, the signals were detected with enhanced chemiluminescence (ECL) kit (New Cell & Molecular Biotech, Suzhou, China) and digitized on Image Quant LAS 4000 mini (GE, Boston, MA, USA). Image quantification was performed using Image J software (Version 1.53a, downloaded from https://imagej.net/Downloads).

After three days of culture with or without TGF‐β1, cells were collected from six‐well plates of 5‐kPa, 30‐kPa PGS, and plastic by exposure to 5% trypsin/EDTA (Gibco) for 2–3 min at 37℃. After being washed with PBS (HyClone), cells were scraped and their total proteins were extracted in a Radio‐Immunoprecipitation Assay (RIPA) buffer (Fermentas; Thermo Fisher Scientific, Pittsburgh, PA, USA). Bicinchoninic acid (BCA) protein quantitative analysis kit (P0010S; Beyotime, Shanghai, China) was utilized to quantify the protein concentration in each sample. Briefly, the protein samples were loaded on pre‐cast 10% SDS‐PAGE (PG112; Epizyme, Shanghai, China) and transferred to polyvinyl difluoride (PVDF) membranes (Bio‐Rad). The membranes were incubated at 4℃ overnight with the primary antibodies, which are listed in Table 4, and then incubated with HRP‐labeled secondary antibodies (Arigo; Hsinchu, Taiwan, China) for 1 h at room temperature. Then, the band images were developed with enhanced chemiluminescent (ECL) reagents (NCM Biotech; Suzhou, Jiangsu, China) and digitized on Image Quant LAS 4000 mini (GE Healthcare, Marburg, MA, USA). Image quantification was performed with Quantity One software (Bio‐Rad). GAPDH was used to serve as a loading control. All data were expressed as fold change in protein expression relative to the designated control group.