Anti ephb4

Total proteins in HUVECs were extracted with radioimmunoprecipitation assay (RIPA) b00uffer (Beyotime, Shanghai, China) containing phenylmethylsulfonyl fluoride (PMSF) (1 mM) (Sigma-Aldrich) and quantified using a bicinchoninic acid (BCA) kit (Beyotime). Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The resulting membranes were probed with primary antibodies, followed by the corresponding secondary antibodies. The primary antibodies used were anti-EFNB2 (1:1,000) (SAB, Greenbelt, Maryland), anti-GJA4 (1:1,000) (SAB), anti-EPHB4 (1:800) (Cell Signaling, Boston, MA), anti-MYC (1:1,000) (Cell Signaling), anti-EYA3 (1:1,000) (Proteintech, Wuhan, China), anti-CDK2 (1:1,000) (Proteintech), anti-RRM2 (1:1,000) (Proteintech), anti-MYC (pT58) (1:1,000) (SAB), anti-MYC (pS62) (1:1,000) (SAB), and anti-β-ACTIN (1:5,000) (Proteintech). The secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG (1:5,000) (Proteintech). The bands were detected using a chemiluminescence system (Tanon, Shanghai, China). Quantification was performed using ImageJ2 (Rawak Software, Stuttgart, Germany).

Cells were lysed with Protein Extraction Reagent (Pro-Prep, iNtRON Biotechnology, Seongnam, Korea) and the lysates were centrifuged at 13000 rpm at 4℃ for 15 min. Protein concentrations were determined using the Bradford assay. Equal amounts of proteins were electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose membranes at 100 V for 2 h. The membranes were blocked with 5% milk for 1 h. The membranes were incubated overnight at 4℃ with 1:1000 dilution of anti-EphB2 (Cell Signaling Technology, Danvers, MA, USA), anti-EphB3 (Thermo Fisher Scientific, Inc., Waltham, MA, USA), anti-EphB4 (Cell Signaling Technology), and 1:10000 dilution of anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then, the membranes were incubated with anti-rabbit 1:5000 or anti-mouse 1:5000 (Santa Cruz Biotechnology) secondary antibodies for 1 h at room temperature. After incubation, the protein bands were observed using ECL reagent (iNtRON Biotechnology). Protein expression was quantified using Quantity One® 1-D Analysis Software (Bio-Rad Laboratories, Hercules, CA, USA).