Ripa lysis buffer

Proteins were extracted from the colon tissues using RIPA Lysis Buffer (Wuhan Servicebio Technology Co., Ltd, China) and the protein concentration was determined by bicinchoninic acid (BAC) method on the enzyme labelling instrument (Thermo Fisher Scientific, USA). The SDS-PAGE was used to separate the protein, and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were first blocked for 1h with 5% BSA at room temperature, and then probed with primary antibodies of MMP1 (Affinity, dilution of 1:1000), MMP3 (Abcam, dilution of 1:5000), MMP7 (Abcam, dilution of 1:1000), MMP9 (Abcam, dilution of 1:1000), MMP12 (Proteintech, dilution of 1:1000) and β-Actin (Affinity, dilution of 1:5000) at 4°C for overnight and then incubated with HPR-conjugated antibody at 37°C for one hour. Finally, the protein bands were recorded by ELC (Thermo Fish Scientific, USA) and quantified by Image J software to evaluate the protein relative expressions compare with β-Actin.

Following transfection, total proteins were extracted with RIPA lysis buffer (Wuhan Servicebio Technology Co., Ltd.) containing protease/phosphatase inhibitor cocktail. The total protein concentration was measured with a BCA protein assay kit (Beijing Solarbio Science and Technology Co., Ltd.). Equal amounts of protein per lane (25 µg) were separated by SDS-PAGE on 10% gels and were transferred onto PVDF membranes. Membranes were blocked with 5% skimmed milk for 1 h at room temperature and probed with primary antibodies at 4°C overnight. The primary antibodies included anti-CDC25A (cat. no. ab989; 1:1,000 dilution; Abcam), anti-Ki67 (cat. no. ab16667; 1:1,000; Abcam), anti-PCNA (cat. no. ab92552; 1:1,000; Abcam) and anti-E2F1 (cat. no. ab288369; 1:1,000; Abcam) antibodies. Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (cat. no. ab6721; 1:2,000; Abcam) at room temperature for 2 h and images were acquired using a Tanon-5200 Chemiluminescence Imager (Tanon Science and Technology Co., Ltd.). Finally, the band density was analyzed using ImageJ software v1.8.0 (National Institutes of Health).

Total protein was extracted from H9c2 cells using RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors (Wuhan Servicebio Technology Co., Ltd.). Protein concentration was determined using the BCA protein assay kit (Wuhan Servicebio Technology Co., Ltd.). Proteins (15 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gels and were transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk in 1X TBS-0.5% Tween 20 (Biosharp Life Sciences) for 1 h at room temperature. The blots were then incubated with primary antibodies against RICH1 (1:500; cat. no. sc-514438; Santa Cruz Biotechnology, Inc.) and α-tubulin (1:2,000; cat. no. GB15201; Wuhan Servicebio Technology Co., Ltd.) overnight at 4°C, followed by anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:50,000; cat. no. BL001A; Biosharp Life Sciences) or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:50,000; cat. no. BL003A; Biosharp Life Sciences) for 1 h at room temperature. Protein bands were visualized using a Chemiluminescence Kit (Biosharp Life Sciences). Bio-ID software (Version 6.0; Bio-Rad Laboratories, Inc.) was used to semi-quantify protein expression.