Bicinchoninic acid quantitative kit

Cells were lysed for 30 min on ice, centrifuged at 4°C for 15 min, and the supernatant was discarded. The total protein in each group was measured using a bicinchoninic acid quantitative kit (Beyotime Biotech Inc). Protein samples were separated using sodium lauryl sulfate‐polyacrylamide gel electrophoresis. The separated proteins were transferred to a polyvinylidene difluoride membrane, incubated with 3% bovine serum albumin at room temperature, and then incubated with primary antibodies. After incubating overnight at 4°C, the membrane was washed five times with tris‐buffered saline–Tween 20 (TBST) every 5 min. After 2‐h incubation with the secondary antibody (1:8000) at room temperature, the membrane was washed three times with TBST every 5 min. Following this, electrochemiluminescence imaging was performed.
The experiment was performed with commercial antibodies, RCC1 (Proteintech, 22142‐1‐AP), MIB1 (Proteintech, 11893‐1‐AP), Notch1 (D6F11, Cell Signalling Technology), Notch2 (Bioss, bs‐21664R), and Notch3 (Proteintech, 55,114‐1‐AP).

The cells were collected and disrupted by sonication, total protein was extracted, protein concentrations were determined using a bicinchoninic acid quantitative kit (Beyotime Biotechnology, China), and sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% non-fat dry milk at 4°C for 6 h. Mouse anti-human UNC13B monoclonal antibody (ab97664, abcam) was added to the membrane and incubated overnight at 4°C. Mouse or goat anti-human IgG secondary antibody was then incubated for 2 h. The contents of UNC13B, MAP3K7 (ab109526, abcam), CDK4 (#12790, CST), and PINK1 (ab23707, abcam) proteins on PVDF membranes were detected by chemiluminescence.