Phenyl methyl sulfonyl fluoride (pmsf)

For SDS PAGE, cells were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with protease inhibitor phenylmethylsulfonyl fluoride (PMSF, 1mM, Sangon Biotech, Shanghai, China), and heated to 95°C for 5 min. Blue native PAGE was performed according to a previously described protocol (29 (link)). For blue native PAGE, cells were lysed in solubilization buffer (50 mM NaCl, 50mM imidazole, 2 mM 6-aminohexonic acid, 1mM EDTA, pH 7.00) and supplemented with 2.5% digitonin (w/v, Sigma-Aldrich) and 1mM PMSF (Sangon Biotech). Proteins resolved by SDS-PAGE or blue native PAGE were transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes were then immunoblotted with primary antibodies and secondary antibodies. Primary and secondary antibodies was shown in Supplementary Table 1.

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.

Cells were lysed with RIPA lysis buffer and Phenylmethyl sulfonyl fluoride (PMSF, A610425, Sangon Biotech). The protein concentration was determined by a BCA protein assay kit (Thermo). Equal amount of protein samples in SDS sample buffer (1% SDS, 62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 5% mercaptoethanol, and 0.05% bromphenol blue) were boiled for 5min and subjected to reducing 10% SDS-PAGE. Proteins were separated by SDS-PAGE and electrophoretically transferred to PVDF membrane. The membrane was blocked with 5% non-fat milk powder in PBST at room temperature for 2h, and then incubated with primary antibodies at 4℃ overnight. Following washing, the membrane was incubated with secondary antibody conjugated with horseradish peroxidase (anti-rabbit or anti-mouse) at room temperature. The following antibodies were used: antibodies against phosphor-MAPKAPK-2 (Thr334) (#3041, 1:1000), and AKT (9272S, 1:1000) were purchased from Cell Signaling Technology; antibodies against AKT (phospho-ser473) (11054, 1:1000), ERK1/2 (phospho-Thr202) (12082, 1:1000) and ERK1/2 (29162, 1:1000) were purchased from Signalway Antibody; antibody against V5-tag (66007-1-Ig, 1:1000) was purchased from Proteintech.