Bca protein assays kit

Total protein was isolated from the primary cardiomyocytes using RIPA buffer (Beyotime Biotechnology, Shanghai, China) with the protease inhibitor cocktail. Protein concentration was quantified with BCA protein assays kit (Beyotime Biotechnology, Shanghai, China). 30-70 μg of proteins was separated by SDS-PAGE gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked by 5% nonfat dry milk with PBS containing 0.1% Tween-20 at room temperature for 1 h and then incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase- (HRP-) conjugated secondary antibodies at room temperature for 1 h. The primary antibodies used in this study were listed as follows: anti-Rap1GAP (1 : 10000, Abcam, Cambridge, UK), anti-LC3B I/II (1 : 1000; Cell Signaling Technology, MA, USA), anti-P62 (1 : 1000; Cell Signaling Technology), anti-Akt and anti-p-Akt (1 : 1000; Cell Signaling Technology), anti-mTOR and anti-p-mTOR (1 : 1000; Cell Signaling Technology), anti-P70s6k and anti-p-P70s6K (1 : 1000; Cell Signaling Technology), and GAPDH (1 : 1000; Cell Signaling Technology). Protein levels were normalized to GAPDH, and the results of signals were quantified by the software of ImageJ.

CRC cells and macrophages were lysed by the RIPA solution to extract the protein (Thermo Fisher Scientific, Waltham, MA, USA), then the protein concentration was measured with the BCA Protein Assays Kit (Beyotime, Shanghai, China). The proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane, which was then blocked for 2 h at room temperature, and subsequently incubated with primary antibodies, including anti-GAPDH (5174, 1:1000, Cell Signal Technology, Danvers, MA, USA), anti-TGF-β1 (ab215715, 1:1000, Abcam, Cambridge, UK), and anti-CST3 (ab109508, 1:1000, Abcam, UK) overnight at 4 °C. Next, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (7076, 1:1000, Cell Signal Technology, Danvers, MA, USA) for 2 h at room temperature. Blots were visualized by enhanced chemiluminescence on a Tanon 5200 system (Tanon, Shanghai, China).