Bicinchoninic acid protein concentration kit

Protein levels were measured as previously described [24 ]. At 48 h post-transfection, 5–8 F and SUNE-1 cells were lysed using the radioimmunoprecipitation assay buffer (lysis buffer; Abcam, China). We quantified the supernatants using a bicinchoninic acid protein concentration kit (Beyotime, China). Subsequently, proteins (20 µg) were separated with 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and were electro-transferred onto a polyvinylidene fluoride membrane. The membrane was incubated with 5% skimmed milk for 1 h at room temperature before being incubated with rat anti-CSE1L (1:1000, Cat. no. ABIN560494, Beijing 4A Biotech Co, Ltd., China), and rat anti-GAPDH (1:1000, Cat. no. AF1186, Beyotime, China) at 4°C for 24 h. The following day, the blot was probed with a mouse anti-rabbit horseradish peroxidase–conjugated secondary antibody (1:1000, Cat. no. 4060–01, Southern Biotech, USA), and protein bands were visualized by chemiluminescence using ECL (Beyotime, China).

Protein was extracted from the myocardium around the infarcted area (Solarbio, China), and the concentration was measured using a bicinchoninic acid protein concentration kit (Beyotime, China). The protein samples were separated by 10% acrylamide gel electrophoresis and transferred to nitrocellulose membranes, which were later incubated in the first antibody at 4°C overnight. The following target protein was detected to obtain their expression level: Jagged-1 (ab109536, Abcam, United Kingdom), Notch1 (3608, CST, United States), NICD (ab52301, Abcam, United Kingdom), Hes-1 (11988, CST, United States), GAPDH (Hangzhou Good Here Biotechnology, China). After the membranes were washed three times with PBS-T (PBS containing 0.5% Tween 20), they were further incubated in the secondary antibody (Jackson ImmunoResearch, United States) for 1 h at room temperature away from light. Then, bands were acquired using ECL visualization. The images were analyzed by the Image-J software.