Anti eya3

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).

Total proteins were extracted using RIPA buffer containing a protease inhibitor cocktail. After centrifuging at 13,000 rpm for 15 minutes at 4 ℃, the soluble cell extracts were used for immunoblots according to a previous protocol (10 (link)). The primary antibodies included the following: anti-EYA3 (Proteintech, Wuhan, Hubei, China; #21196-1-AP), anti-SIX5 (Invitrogen, Shanghai, China; #PA5-75417), anti-p300 (Santa Cruz Biotechnology, Shanghai, China; sc-48343), anti-GAPDH (Santa Cruz Biotechnology; #sc-47724), anti-Flag (Sigma-Aldrich; #SAB4200071), and anti-Myc (Invitrogen; #R95125). The horseradish peroxidase (HRP)-conjugated secondary antibodies included the following: anti-mouse immunoglobulin G (IgG) (Sigma-Aldrich; #GENA931) and HRP-conjugated anti-rabbit IgG (Sigma-Aldrich; #GENA934).