3.3.7. Western Blotting on HUVEC Cells—VEGF Stimulated

HUVEC were seeded in a 6-well plate pre-coated with 0.2% gelatin and Collagen G at 0.1 mg/mL in PBS. The cells were cultured in M199 (GIBCO) supplemented with 10% fetal calf serum, 1% Endothelial Cell Growth Supplement (EmdMillipore), 0.1 mg/mL of heparin sodium, 0.1 mM of hydrocortisone (Sigma Aldrich, Milan, Italy), 1% antibiotic glutamine mixture and 0.1% of vitamin C for 48 h to become confluent. Confluent HUVEC were starved for at least 4 h with the medium alone before stimulation. After starvation, the cells were pre-incubated with the indicated compound at 20 μM for 10 min at 37 °C and then stimulated with both VEGF at 50 ng/mL and the compound at 20 mM for 20 min. As the compounds were dissolved in dimethylsulfoxyde (DMSO), incubation with DMSO was used as a control to the compounds. Treated cells were washed and lysed for protein extraction. Protein concentration was determined with the MicroBCA™ Protein Assay Kit (Thermo Scientific, Waltham, MA USA). Equal amounts of proteins (30 mg) were subjected to a gel electrophoresis and then transferred to nitrocellulose blotting membranes. Membranes were blocked with a blocking buffer made of PBS containing 5% not-fat dry milk and 0.05% of Tween 20. Membranes were incubated overnight at 4 °C with primary antibodies diluted in the blocking buffer. The following primary antibodies were used in this study: rabbit anti-phospho-p38MAPK (pp38MAPK) used at 1:1000 dilution, rabbit anti-phospho-ERK1/2 (pERK1/2) (cell signaling) at 1:1000 dilution, rabbit anti-phospho-AKT (pAKT) at 1:1000 dilution and mouse–tubuline antibody (Millipore) at 1:4000 dilution. Membranes were washed three times for 5 min with PBS-Tween 20 0.05% and incubated at room temperature for 1 h with the adequate secondary antibody. Horseradish peroxidase (HRP)-coupled goat anti-rabbit antibody was used at a dilution of 1:10000 and the HRP-goat anti-mouse antibody at 1:3000 dilution. Both secondary antibodies were from Jackson ImmunoResearch. Membranes were washed and the signals detected using the enhanced chemioluminescence system (Advansta, WesternBright™ Sirius). Protein band intensities were quantified with ImageJ and the tubuline intensity was used to ensure equal loaded protein amounts. The results were expressed relative to the condition of treatment with VEGF and DMSO serving as control in the same blot and set at 100%. Data are representative of at least two independent experiments.