Horseradish peroxidase hrp coupled antibodies

For immunofluorescence and immunoblot analyses, we used Alexa-coupled antibodies from Invitrogen and horseradish peroxidase (HRP)-coupled antibodies from Jackson ImmunoResearch Laboratories (West Grove, PA, USA), respectively. Primary antibodies used were rabbit anti-Rap1 (recognizing both Rap1a and Rap1b; Cell Signaling, Danvers, MA, USA), goat anti-VE-cadherin and rabbit anti-Tie2 from Santa Cruz Biotechnology (Santa Cruz, CA, USA), mouse anti-phosphotyrosine (clone 4G10; Millipore Sigma, Burlington, MA, USA), and anti-β-actin (Cell Signaling) and mouse anti-eNOS (BD Biosciences, San Jose, CA, USA).
For immunoblotting, cells were solubilized with a lysis buffer containing 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 0.1% deoxycholic acid, 50 mM Tris-HCl (pH 7.4), 0.1 mM EGTA, 0.1 mM EDTA, 20 mM sodium fluoride, 1 mM sodium pyrophosphate tetrabasic, and 1 mM sodium orthovanadate. Lysate were incubated for 30 min at 4 °C, centrifuged at 14,000× g for 10 min, boiled in SDS sample buffer, separated by SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane (Hybond-ECL; GE Life Sciences, Pittsburg, PA, USA), and Western-blotted. Antibody detection was performed with HRP-coupled antibodies from Jackson Laboratories and using the Image Quant LAS4000 chemiluminescence-based detection system (enhanced chemiluminescence; GE Life Sciences).