VEGF-A and FGF-2 Stimulation of HMEC-1 Cells

HMEC-1 were grown in 6-well titre plates. Cells were serum-arrested at ~80%–90% confluency for 24 h. Cells were then treated with TRAIL (10 ng/mL), VEGF-A (10 ng/mL) and FGF-2 (10 ng/mL) for 15 min, followed by total protein isolation using 1× SDS-sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 50 mM dithiothreitol and 0.1% bromophenol blue), as previously described [4 (link)]. Lysates were boiled for 5 min, followed by separation using 10-well Bolt™ 4%–12% Bis-Tris Plus Gels (Thermo Fisher Scientific, Waltham, MA, USA). The iBlot Gel Transfer System (Life Technologies, Carlsbad, CA, USA) was used to transfer proteins onto nitrocellulose membranes. Membranes were blocked in 5% bovine-serum albumin (in 1× PBS-Tween-20) for 1 h and then incubated overnight at 4 °C with rabbit polyclonal phospho-p42/44 MAPK(ERK1/2) antibody (1:5000; Cell Signaling Technology, Danvers, MA, USA) or 42/44 MAPK(ERK1/2) (1:3000; Cell Signaling Technology, Danvers, MA, USA). Proteins were detected with horseradish peroxidase-conjugated secondary anti-rabbit antibodies (1:3000; Dako, Glostrup, Denmark) and visualized by chemiluminescence (Amersham, GE Healthcare Life Sciences, Logan, UT, USA).