Anti ephb4

Cells were lysed with radioimmunoprecipitation assay buffer (RIPA) buffer (Beyotime Institute of Biotechnology) and 1 mM phenylmethylsulfonyl fluoride (PMSF) (Beyotime Institute of Biotechnology). Forty micrograms of protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred onto nitrocellulose membranes (Pall Corp., Pensacola, FL, USA). Blocked in 5% (w/v) milk for 1 h, membranes were then incubated with the following primary antibodies overnight at 4°C: anti-ephrinB2 (1:2000; Abcam, Cambridge, UK), anti-phospho-ephrinB2 (Tyr324/329; 1:500; Cell Signaling Technology, Danvers, MA, USA), anti-EphB4 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-EphB4 (1:1000; Signalway Antibody, College Park, MD, USA), and anti-β-actin (1:3000, Beyotime Institute of Biotechnology). Blots were then incubated with horseradish peroxidase–conjugated goat anti-rabbit or anti-mouse secondary antibodies (Proteintech, Wuhan, Hubei, China). Tanon4500 Immunodetection System (Tanon Science & Technology Co., Ltd., Shanghai, China) was used for visualization of blots. Quantification of the blots was performed using Image J.

Cells grown in glass slides were treated with shear stress for 2 h. Then total protein was obtained by using M-PER protein extraction buffer, and quantified using a BCA kit (Thermo Fisher Scientific, Inc.) and separated on 7.5 or 12% polyacrylamide gel followed by transfer onto an ImmunBlot PVDF membrane (GE Healthcare Life Sciences, Little Chalfont, UK). The membranes were blocked for 1 h with 5% BSA in Tris-phosphate buffer containing 0.05% Tween-20 (TBS-T). It was further incubated overnight at 4°C with anti-Notch1 (1:2,000; ab52627), anti-DLL4 (1:1,000; ab7280), anti-Hey1 (2 μg/ml, ab22614), anti-Hey2 (2 μg/ml; ab25404) all from Abcam (MA, USA); anti-EphrinB2 (1:1,000; sc-398735) and anti-EphB4 (1:1,000; sc-130081) both from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); anti-VEGFR2 (1:2,000; ab39256) and anti-CD31 (1:500; ab28364) both from Abcam, primary antibodies. After three washes (5 min) with TBS-T, membranes were further incubated with HRP-conjugated secondary antibodies: anti-rabbit (cat. no. 7074) or anti-mouse (cat. no. 7076) both from Cell Signaling Technology, Inc., (Danvers, MA, USA) for 1-2 h and followed by three washes with TBS-T. The target protein signal was detected and digitized using ECL (Thermo Fisher Scientific, Inc.).