Fig. 2: Measurement of ligand-independent and dependent VEGFR activation on the plasma membrane.
From: Molecular basis of VEGFR1 autoinhibition at the plasma membrane
a–d Confocal images of VEGFR2 or VEGFR1 fused to mCherry in a low (a, c) and high (b, d) expressing CHO cell lines. The VEGFR expression level is shown in red (λex = 552 nm, λem = 586-651 nm), and the phosphorylation status is shown in green (λex = 488, λem = 505-531). Scale bar = 10 μm. e, f The expression level of VEGFR2 (panel e) or VEGFR1 (panel f) is plotted against the phosphorylation level of the corresponding tyrosine residues at the C-terminal tail. The low-expressing and high-expressing cells are indicated based on the mCherry intensity at the plasma membrane. Individual data points in the left panel represent the mean expression and phosphorylation level for the binned cells. The orange line represents the linear fitting of the individual data points in the ligand-dependent activation. The blue line in panel e represents the second-order polynomial fitting of the individual data points in the ligand-independent activation. In panel f, the blue line is the guiding line. The right panel represents the bar plot of the normalized phosphotyrosine levels. The phosphotyrosine level (FITC channel) is normalized with respect to the corresponding VEGFR expression level (mCherry channel) at the plasma membrane. In (e) (left), n = 85 (VEGFR2-VEGF165), 89 (VEGFR2 + VEGF165), and in (f) (left) n = 107 (VEGFR1-VEGF165), 100 (VEGFR1 + VEGF165) cells were examined over five independent experiments in (e, f) (right) Each bar represents the mean value of 30–40 cells in the bar plot. The error bar shows the standard deviation of data points. Data are presented as mean values ± SD from five independent experiments. g The immunoblot shows the representative phosphorylation level of VEGFR1 or VEGFR2 at the indicated time points after activating the transfected CHO cell line with 50 nM VEGF165. (n = 3). h The plot of the phosphorylation level of respective C-terminal tyrosine residue as a function of time. The phosphorylation level is analyzed from the densitometric measurement of the Western blot shown in (g). The t1/2 is determined by fitting the decay of the highest intensity observed to exponential decay. Data are presented as mean values ± SD from three independent experiments. All data were plotted using GraphPad Prism Ver 9.5.1. The confocal images were generated using Fiji Ver 1.54 f. The schematics were made using Inkscape Ver 1.2. Source data are provided as a Source Data file for panels e-h. See Supplementary Figs. 1 and 2.
