Bca protein kit

Intestinal tissues were homogenized in a protease inhibitor cocktail containing radioimmunoprecipitation assay (RIPA) lysis buffer (Boster, Wuhan, China). The protein concentration was quantitatively determined using bicinchoninic acid (BCA) protein kit (Boster). The extracted protein was separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk and incubated with primary antibodies (including PFKFB3, NLRP3, caspase-1, GSDMD, IL-18, and IL-1β) (1 : 1000, ab, Abcam, USA) overnight at 4°C. After that, the membranes were incubated with rabbit HRP-conjugated secondary antibody at room temperature for 2 h, and the protein bands were visualized using chemiluminescent peroxidase substrate (Millipore, Boston, MA, USA). GAPDH antibody was used as the internal control. Densitometric analysis was quantified using the ImageJ software (National Institutes of Health, USA).

Total RNA was isolated using RNA extraction kit (Takara Bio, China) and qPCR was performed. The following primers were used: VEGF A (sense); 5′- CAA​AGC​CAG​CAC​ATA​GGA​GAG​A-3′, and VEGF B (antisense); 5′-CTA​TCT​TTC​TTT​GGT​CTG​CAT​TCA​C-3′; rat actin A (sense); 5′-CCC​ATC​TAT​GAG​GGT​TAC​GC-3′, and rat actin B (antisense); 5′-TTT​AAT​GTC​ACG​CAC​GAT TTC-3’ (Takara Bio, China). EPC protein was extracted using cell lysis buffer (Beyotime, China) and VEGF protein expression was determined using western blotting. Total cell protein was extracted using RIPA buffer containing phosphatase and protease inhibitors (PMSF) (Beyotime, China). Protein concentration in the supernatant was measured using a BCA protein kit (Boster Bio, China). Protein was separated using SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) (Millipore, United States) membrane. The membrane was incubated overnight at 4°C with primary antibodies against VEGF (1:2,000) and β-actin (1:1,000) (Abcam, United States). The PVDF membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000) for 30 min. The membranes were washed again, treated with chemiluminescence (ECL) solution (Millipore, United States), and visualized by exposure to film. The expression of each protein relative to β-actin was determined using ImageJ software.