Receptor-Mediated Endocytosis of VEGF-A in Rat Liver Sinusoidal Endothelial Cells
Figure 1
Expression of VEGF receptors in LSECs. (a) Saturation binding of [125I]-VEGF-A to LSECs. LSECs (4 × 106/ml) were incubated with increasing concentrations of [125I]-VEGF-A (0.5-900 pM). After 90 min at 4°C, specific binding was determined as described in “Materials and Methods.” The curve was fitted using nonlinear regression with GraphPad Prism. Data represent the mean ± range of duplicate measurements from a representative experiment performed three times with similar results. (b) Quantification of VEGFR1 and VEGFR2 mRNA expression. After total RNA extraction and cDNA amplification, mRNA levels were quantified by real time RT-PCR as described in “Materials and Methods.” β actin mRNA level was used as internal control for normalization. Data represent the mean ± SD from three independent experiments. No statistical difference was found between the expression levels of VEGFR1 and VEGFR2 using unpaired t-test (p = 0.12). (c) Effect of the VEGFR1-binding peptide on cell surface binding of [125I]-VEGF-A. LSECs (4 × 106/ml) were preincubated at 4°C with the VEGFR1-binding peptide (400 μM in DMSO) or an equal concentration of the control peptide and then incubated with [125I]-VEGF-A. After 90 min at 4°C, specific binding was determined as described in “Materials and Methods.” Data represent the mean ± range from two independent experiments.