HUVEC Protein Signaling Pathways

The protein of HUVEC cells treated or not with 100 nM recombinant vasoinhibin or Vi45–51 followed by the addition or not of 100 ng mL⁻¹ VEGF, was extracted in RIPA lysis buffer supplemented with 1:100 Halt Protease-Phosphatase Inhibitor cocktail and 5 mM EDTA (both from Thermo Scientific). Proteins were resolved in SDS-PAGE followed by western blot as reported [25 (link)] with antibodies against phospho-ERK1/2 (9101, 1:500), phospho-Akt (9271, 1:500), or phospho-eNOS (9571, 1:250); and antibodies against total proteins ERK1/2 (9102, 1:500), Akt (9272, 1:500), or eNOS (9572, 1:500), all from Cell Signaling (Danvers, MA).