Mouse anti notch1

Six rats per group were used for western blot detection at the corresponding sampling time. Before protein cleavage, PMSF (Sangon Biotech) was added to RIPA Lysis Buffer I (Sangon Biotech), lysed on ice for 1 h, and the supernatant was collected. After that, the protein concentration was measured by the BCA method. Absorb 50 g of protein per sample's concentration, mix with loading buffer in a 4:1 ratio, and dilute to 20 L. Fill it up with ddH2O. The concentrated gel and separation gel electrophoresis voltages are 80 V and 100 V. Electrophoresis is complete when bromophenol blue reaches the bottom of the gel. The PVDF membrane was blocked in 5% skim milk for 2 h after the protein sample was transferred. After blocking, the membrane was gently washed with TBST, rabbit anti‐VEGFA (1:100, Santa Cruz Biotechnology), mouse anti‐Notch1 (1:100, Santa Cruz Biotechnology), and rabbit anti‐Hes1 (1:100, Santa Cruz Biotechnology). Antibodies were incubated overnight at 4°C. After 16 h, the membrane was taken out and washed on the TBST shaker for 3−5 min. After washing, add anti‐HRP goat anti‐mouse IgG‐Fc II (Cell Signaling Technology) and shake for 1 h. Darkroom imaging with ECL color developing solution. ImageJ software was used to measure the gray value of VEGFA, Notch1, Hes1, and ‐actin proteins. Finally, the final relative expression was compared according to the ratio.

Tissues were homogenized in RIPA plus buffer containing a cocktail of EDTA-free protease inhibitors. The homogenate was centrifuged at 12,000 rpm for 30 min at 4°C. Protein concentration was assayed using the BCA method, then loaded and subjected to electrophoresis in 10% SDS-PAGE gels, and transferred onto PVDF membranes. The membranes were then blocked in 5% BSA for 2 hour and then incubated with one of the following primary antibodies: rabbit anti-caspase-3 (1 : 500, Proteintech) or mouse anti-Bax (1 : 100, Proteintech) or rabbit anti-Bcl-2 (1 : 500, Proteintech) or mouse anti-Notch1(1 : 500, Santa Cruz) or mouse anti-Hes1 (1 : 1000, Santa Cruz), with gentle shaking at 4°C overnight. Then, horseradish peroxidase conjugated goat anti-mouse IgG (1 : 50000, Proteintech) or goat anti-rabbit IgG (1 : 50000, Proteintech) secondary antibodies were incubated with the membranes for 2 hours at room temperature. The immunopositive bands were visualized using an enhanced chemiluminescent substrate (Thermo Fisher) and Bio-Rad ChemiDoc XRS digital documentation system. The amount of protein expression is presented relative to the levels of β-actin.