Beyoeclplus chemiluminescence reagent

Cultured cells (5 × 10⁶) were lysed on ice using PMSF lysate buffer. Samples containing 40–60 μg of protein were subjected to thermal denaturation and SDS-PAGE and were then transferred to PVDF membranes. After the membrane was sealed with a blocking solution (TBST containing 5% skim milk) for 60 min, it was incubated with the primary antibody: anti-ERG (Abcam, England, Ca#133264), anti-VE-cadherin (Abcam, Ca# ab33168), and anti-claudin-5 (Abcam, Ca# ab15106) overnight and then with the HRP-labeled secondary antibody (Proteintech, USA, Ca# SA00001-1) at 37°C or 1 h. Finally, the BeyoECLPlus chemiluminescence reagent (Beyotime, China, P0018) was used to visualize the protein bands.

The proteins from the cultured cells were harvested by using a phenylmethylsulfonyl fluoride (PMSF) lysate buffer. Samples containing 50 μg of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to polyvinylidene fluoride membranes. After the membranes were blocked with 5% non-fat milk for 1 h, they were incubated with the primary antibody at 4 °C overnight: NEIL3 (ab230908; Antibody Cambridge (Abcam)), p-ATR (2853S; Cell Signaling Technology (CST)), p-ATM (5883S; CST), γ-H2AX (9718S; CST), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118S; CST). The membranes were then incubated with the horseradish peroxidase (HRP)-labeled secondary antibody (Proteintech, Wuhan, Hubei, China) at 37 °C for 1 h. The protein band signal was detected using the BeyoECLPlus chemiluminescence reagent (Beyotime, Shanghai, China).