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Fig. 3 | Cell Communication and Signaling

Fig. 3

From: Recombinant RGD-disintegrin DisBa-01 blocks integrin αvβ3 and impairs VEGF signaling in endothelial cells

Fig. 3

DisBa-01 decreases VEGFR2 protein content. a Analysis of VEGFR2 protein content by western blot. HUVECs (5 × 105 cells/well) were seeded in 6-well plates and left to adhere on an incubator at 37 °C, 5% CO2, overnight, followed by a period of 24 h of starvation at serum-free medium. Cells were treated with 1 ml of DMEM supplemented with 10% FBS and either DisBa-01 (1000 nM), VEGF (10 ng/mL) or a co-treatment and incubated for 1 and 24 h at 37 °C, 5% CO2, followed by cell lysis. Twenty micrograms of protein from cell lysates were separated on SDS-PAGE. Blots were probed with VEGFR2 antibody and GAPDH antibody was used to normalize analysis. Bands corresponding to all proteins were quantified by densitometry using the ImageJ FIJI program. Bar graph shows the mean ± SE of VEGFR2/GAPDH expression from three independent experiments. b VEGFR2 mRNA (KDR) expression. HUVECs (5 × 105/well) were seeded in 6-well plates containing DMEM and 10% FBS, followed by a 24-h starvation period on serum-free medium. Cells were treated with DisBa-01 (1000 nM) and/or VEGF (10 ng/mL) for 24 h followed by lysis and RNA isolation. Quantitative RT-PCR was carried out using specific primers to human KDR (VEGFR2) and GAPDH (housekeeping). Bar graph shows the mean ± SE of VEGFR2 expression from three independent experiments. Values of *p < 0.05 were significantly different when compared to untreated (a), DisBa-01 (b) and VEGF (c) groups

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