American Society of Hematology

Figure 1

$Figure 1. VEGF stimulates exit of VEGFR2 from the trans-Golgi complex. (A) Serum-starved HUVECs were labeled with mAb against VEGFR2 (55B11) and TGN46 (Golgi marker). Total cell-associated and Golgi-localized fluorescence intensity of VEGFR2 was quantified by image analysis. Values are expressed as a fraction of the total VEGFR2 in the Golgi apparatus. (B) Homogenates prepared from serum-starved HUVECs were fractionated on a self-generated Optiprep gradient (10%, 20%, 30%) and immunoblotted with antibodies against proteins enriched in the PM (vascular endothelial-Cadherin); the trans-Golgi complex (TGN46); or endosomes (EEA1). (C) Percentage of total VEGFR2 and TGN46 in each fraction, based on quantification of density of bands in each fraction obtained by Optiprep gradient centrifugation. (D) Effects of VEGF-A treatment on VEGFR2 localization at the Golgi apparatus. Serum-starved HUVECs were treated with CHX (10 μg/mL), and immunofluorescence imaging was carried out for VEGFR2 and TGN46 localization. (E) Quantification of the Golgi-localized VEGFR2 (overlapping with TGN46) shown in panel D. Values are expressed as a percentage of change in intensity of Golgi-localized VEGFR2 signal (relative to initial intensity in at 0 minutes chase at 37°C, data not shown). Percentages in panels A and E represent mean (± SD) in n = 90 cells for each condition from 5 separate experiments. For panel E, P ≤ .05. (F-G) Effects of BFA treatment on VEGFR2 transport in HUVECs. (F) Representative images of immunofluorescence analysis of untreated and BFA-treated cells stained with VEGFR2 antibody are shown. (G) Biotinylation-based analysis of cell-surface VEGFR2. Surface proteins labeled with the biotinylation reagent sulfo-NHS-SS–biotin were pulled down with streptavidin-Sepharose, and 5% of the total cell lysate and biotinylated cell-surface protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Western blot analysis with antibody against VEGFR2. (H) Quantification of band density for the cell-surface VEGFR2. Percentage is expressed as the change in surface VEGFR2 after BFA treatment (relative to initial levels). The percentage represents mean (± SD) for n = 3 and P ≤ .05. Scale bar represents 5 μm.$

VEGF stimulates exit of VEGFR2 from the trans-Golgi complex. (A) Serum-starved HUVECs were labeled with mAb against VEGFR2 (55B11) and TGN46 (Golgi marker). Total cell-associated and Golgi-localized fluorescence intensity of VEGFR2 was quantified by image analysis. Values are expressed as a fraction of the total VEGFR2 in the Golgi apparatus. (B) Homogenates prepared from serum-starved HUVECs were fractionated on a self-generated Optiprep gradient (10%, 20%, 30%) and immunoblotted with antibodies against proteins enriched in the PM (vascular endothelial-Cadherin); the trans-Golgi complex (TGN46); or endosomes (EEA1). (C) Percentage of total VEGFR2 and TGN46 in each fraction, based on quantification of density of bands in each fraction obtained by Optiprep gradient centrifugation. (D) Effects of VEGF-A treatment on VEGFR2 localization at the Golgi apparatus. Serum-starved HUVECs were treated with CHX (10 μg/mL), and immunofluorescence imaging was carried out for VEGFR2 and TGN46 localization. (E) Quantification of the Golgi-localized VEGFR2 (overlapping with TGN46) shown in panel D. Values are expressed as a percentage of change in intensity of Golgi-localized VEGFR2 signal (relative to initial intensity in at 0 minutes chase at 37°C, data not shown). Percentages in panels A and E represent mean (± SD) in n = 90 cells for each condition from 5 separate experiments. For panel E, P ≤ .05. (F-G) Effects of BFA treatment on VEGFR2 transport in HUVECs. (F) Representative images of immunofluorescence analysis of untreated and BFA-treated cells stained with VEGFR2 antibody are shown. (G) Biotinylation-based analysis of cell-surface VEGFR2. Surface proteins labeled with the biotinylation reagent sulfo-NHS-SS–biotin were pulled down with streptavidin-Sepharose, and 5% of the total cell lysate and biotinylated cell-surface protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Western blot analysis with antibody against VEGFR2. (H) Quantification of band density for the cell-surface VEGFR2. Percentage is expressed as the change in surface VEGFR2 after BFA treatment (relative to initial levels). The percentage represents mean (± SD) for n = 3 and P ≤ .05. Scale bar represents 5 μm.

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