Cell density influences VEGFR-2 expression and activation. (A) Analysis of protein expression levels under different cell density conditions. Equal amounts of whole-cell lysates (WCL) from long-confluent (LC), confluent (C), and sparse (S) ECs were separated by SDS-PAGE and immunoblotted as indicated. (B) Analysis of the surface and intracellular pools of VEGFR-2 in unstimulated LC and S ECs. Surface VEGFR-2 was labeled with the membrane-impermeant sulfo-NHS-SS-biotin, as reported in “Biochemical quantification of VEGFR-2 distribution.” Biotinylated surface VEGFR-2 was collected by binding to streptavidin-agarose. Aliquots of the total cell lysate, surface fraction, and internal fraction were then separated by SDS-PAGE and immunoblotted as indicated. Only the percentages of surface receptors for both LC and S ECs are shown. (C) Quantification of the relative surface and internal pools of VEGFR-2. Values are the mean ± SD of 3 independent experiments. (D-E) Time-course and dose-response analysis for VEGFR-2 phosphorylation. Starved LC and S ECs were left untreated or treated with 2 concentrations of VEGF (0.01 and 0.75nM) at the indicated time points (D) or with increasing VEGF doses (0-1.25nM) for 5 minutes (E). (F) VEGFR-2 activation as a function of VEGF concentration: comparison between experimental data (symbols) from dose-response analysis and model (solid lines, equation 6 in supplemental Section 1.1). Experimental values of phosphorylated Y1054-VEGFR-2 (pY1054-VEGFR-2) were obtained using vinculin signal as normalizer. Values are the mean ± SD of 3 experiments. The experimental values corresponding to pY1054-VEGFR-2 are displayed as the number of surface receptors per cell.