Fig. 6

YULINK mediates VEGF internalization and VEGFR2 trafficking in HUVECs. A, B CTRL- and YULINK-knockdown HUVECs were incubated with VEGF-biotin and anti-VEGF blocking antibody pretreated with VEGF-biotin for the indicated times (5, 15, and 30 min). Cells were then fixed and stained with Alexa-Fluor-594-conjugated streptavidin for analysis by confocal microscopy. A Quantified VEGF intensity in cells at various time points. The average intensity of the VEGF signal per cell was calculated from at least twenty cells for each group. ●, CTRL. ○, shYULINK. ▲, CTRL with anti-VEGF blocking antibody. △, shYULINK with anti-VEGF blocking antibody. B Confocal fluorescence images of internalized VEGF in vector control (upper row) and YULINK-knockdown cells (lower row) at 30 min. Hoechst, cell nucleus staining. C HUVECs were stained for YULINK (green) or VEGFR2 (red). Overlay images demonstrate colocalization of green and red-stained molecules by a shift towards yellow in a Leica confocal system. The percentage of colocalizing pixels was shown in the far right column. Colocalization rate and Pearson correlation coefficient (r) were determined for pixel intensity correlation between the red and green channels. The data reveal a high percentage of colocalizing pixels for YULINK/VEGFR2. D VEGFR2 internalization was characterized in serum-starved HUVECs with VEGF treatment for the indicated times, before being fixed, permeabilized, labeled with anti-YULINK (green) and anti-VEGFR2 (red) antibodies, and visualized using fluorescent microscopy. E Quantification of VEGFR2/YULINK colocalization ratios were analyzed at each time point from panel D (mean ± SD). Data points represent mean ± SD of two replicate experiments with 25 measurements per timepoint. F HUVECs treated with shRNA against YULINK or vector control (CTRL) were incubated with VEGF-biotin for 30 min. Then HRP-conjugated streptavidin and TMB were added and OD₄₅₀ value was measured. G The amount of VEGFR2 at the membrane fraction in the lysate of HUVECs which were treated with shRNA against YULINK or vector control (CTRL) was shown in western blot. Na⁺/K⁺ ATPase a1 was used as an internal control