Ab25404

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.).