Figure 7

Syndecan-1 regulates VEGFR-2 expression. (A) Representative flow-cytometric analysis showing VEGFR-2 expression by (a) MMECs (thin line), MMEC syndecan-1 shRNA (dark line) and the corresponding isotype control antibody (dotted line); (b) BMECs (thin line), AD-FRP h-syndecan-1 BMECs (dark line) and the corresponding isotype control antibody (dotted line); and (c) HUVECs (thin line), AD-FRP h-syndecan-1 HUVECs (dark line) and the corresponding isotype control antibody (dotted lines). The flow-cytometric histograms are representative of three independent experiments with similar results. (B) Syndecan-1 immunoprecipitated with VEGFR-2. The immune complexes were formed by pre-incubation with anti-syndecan-1 and revealed with an antibody to VEGFR-2 (top), or by pre-incubation with anti-VEGFR-2 and revealed with an antibody to syndecan-1 (bottom). The data are from an individual experiment and are representative of two different lines of MMECs. (C) Representative confocal micrographs showing colocalization of VEGFR-2 and syndecan-1 on cellular membrane under the non-permeabilized condition (original magnification, × 630). (D) Representative confocal micrographs showing intracellular expression of VEGFR-2 in permeabilized cells (original magnification, × 630). (E) Representative confocal micrographs showing colocalization between VEGFR-2 and Rab11 in permeabilized cells (original magnification, × 630). (F) Representative confocal micrographs showing VEGFR-2 localization 30 min after stimulation with VEGF (25 ng/ml) (original magnification, × 630). (G) Comparison of VEGFR-2 mRNA expression between MMECs and MMEC syndecan-1 shRNA by qRT-PCR. The normalized expression of the genes with respect to GAPDH was computed for all samples. Values are expressed as fold change with respect to MMECs and are the mean±s.d. of three independent experiments performed in triplicate. Student's t-test was performed (^*P<0.001 MMEC syndecan-1 shRNA versus MMECs).