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Figure 6 | BMC Medicine

Figure 6

From: The cytoprotective drug amifostine modifies both expression and activity of the pro-angiogenic factor VEGF-A

Figure 6

Amifostine inhibits vascular endothelial growth factor (VEGF)R2 expression, VEGF-A binding and signalling in human umbilcal vein endothelial cells (HUVEC). A Amifostine treatment inhibits VEGFR-2 protein expression in HUVEC. After a 24 h-starvation, subconfluent HUVEC were incubated in the presence or absence of 1 mM WR-1065 for indicated period of time and total proteins were collected in an RIPA buffer and resolved on a 7.5% acrylamide SDS-PAGE gel. Proteins were blotted onto a nitrocellulose membrane and probed with a mouse monoclonal antibody against VEGFR-2. Equal loading of proteins was confirmed by β-actin detection. VEGFR-2 migrated at gel positions corresponding to the 150- and 200-kDa forms, as already reported in HUVEC. The blot is representative of three independent experiments. (B) Amifostine treatment impairs VEGF-A binding on HUVEC. HUVEC monolayers (7000 cells/cm2) were allowed to grow for 3 days and then incubated for 12 h in the absence or presence of 1 mM WR-1065. Cells were then exposed to increasing concentrations of 125I-labelled VEGF-A for 2 h at 4°C. The amount of specifically bound iodinated VEGF-A was then determined. Insert represents Scatchard plot corresponding to the saturation curves. The experiment was carried out three times with similar results. (C) Amifostine treatment impairs mitogen activated protein kiinase signalling in HUVEC. Subconfluent HUVEC in serum-free Dulbecco's modified eagle medium were treated with 10 ng/mL VEGF-A for 10 min, alone or together with 1 mM WR-1065. Cytoplasmic proteins were then collected and were resolved on 15% SDS-PAGE. Total (phosphorylated and unphosphorylated) p42/p44 proteins as well as the phosphorylated proteins (P-p42/P-p44) were probed using specific antibodies. Signal was assessed by quantification of chemoluminescence, and ratios between signal intensities of P-p42/P-p44 and total p42/p44 are given beneath the gel. They were normalized to the ratio obtained for untreated cells (VEGF -, WR-1065-), which is set to 1.

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