Ripa lysis buffer 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.

Proteins were extracted from the cells with RIPA Lysis Buffer I (Sangon Biotech), and the concentration was determined according to standard protocols of the BCA Protein Assay Kit (Sangon Biotech). The total protein (30 μg/well) in the supernatant was separated by 10% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% skimmed milk at room temperature for 2 h, the membrane was incubated with primary antibodies against galectin-3 (dilution 1 : 1000; cat. no. ab209344; Abcam, UK), LC3B (dilution 1 : 1000; cat. no. ab192890; Abcam), caspase 3 (dilution 1 : 1000; cat. no. ab32351; Abcam), caspase 9 (dilution 1 : 1000; cat. no. ab32539; Abcam), cytochrome 9 (dilution 1 : 2000; cat. no. ab134909; Abcam), Bax (dilution 1 : 1000; cat. no. ab32503; Abcam), Bcl-2 (dilution 1 : 1500; cat. no. ab32124; Abcam), and GAPDH (dilution 1 : 5000; cat. no. ab8245; Abcam) at 4°C overnight and then incubated with secondary antibodies (dilution 1 : 10,000; cat. no. ab6940; Abcam) for 2 h at room temperature. The immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Sangon Biotech). The blots were semiquantified by ImageJ software (National Institutes of Health).

Exosomes were lysed with RIPA Lysis Buffer I (Sangon Biotech, Cat: C500005) to obtain the total protein. The protein concentration of each sample was determined using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Cat: A30221). Protein (100 µg) from each sample was subjected to SDS-PAGE (4% stacking and 10% separating gels) and then transferred overnight onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). The primary antibodies used here included anti-human TJP1 antibody (Abcam, Cat: AB216880), anti-CDH2 antibody (Abcam, Cat: AB76057), anti-VIM antibody (Abcam, Cat: AB 137321), anti-EPS15 antibody (Abcam, Cat: AB224811), anti-GAPDH antibody (Abcam, Cat: AB8245), and anti-Flag antibody (Abcam, Cat: AB1162).