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Blood, Vol. 108, Issue 4, 1243-1250, August 15, 2006
Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration
Blood Favier et al.
108: 1243
Supplemental materials for: Favier et al
Files in this Data Supplement:
- Figure S1. Interaction of NRP2-Flag with VEGFR-2 and VEGFR-3-HA in HEK cells (PDF, 46.3 KB) -
(A) HEK293T cells were co-transfected with VEGFR2 and NRP2 Flag. B) Cells were transfected with VEGFR3-HA and NRP2-Flag. Twenty-four hours after transfection, cells were washed and starved overnight in a serum-free medium supplemented with 0.01mg/ml BSA. Cells were stimulated or not for 10 minutes at 37°C, with the indicated ligand. After washing with ice-cold PBS, cells were resuspended in lysis buffer containing 150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1% Triton100, 100 µM Na3VO4 and protease inhibitor cocktail (Sigma-Aldrich). Supernatants were collected and subjected to immunoprecipitation. Flag-tagged NRP-2 was immunoprecipitated with Anti-Flag-agarose (Sigma-Aldrich). Immunoprecipitation of VEGFR-3 was done using anti-HA—conjugated beads (Sigma). Immunoprecipitates were washed five times with 1 ml of lysis buffer. Proteins were eluted by incubation with sample buffer and subjected to western blot analysis using the indicated antibodies. Results confirmed that VEGA and VEGFC induce the heterodimerization between NRP2 and VEGFR-2. However, only VEGF-C was able to induce an heterodimerization with VEGFR-3. It is noteworthy that immunoprecipitation of the complex with either an anti-NRP2 (Figure 1) or an anti-VEGFR3-specific antibodies give rise to exactly the same profile.
- Figure S2. Phenotypic characterization of HMVEC (PDF, 73.3 KB) -
(A) Cell surface expression of VEGFR-1, VEGFR-2 and VEGFR-3 was measured by FACS analysis using monoclonal phycoerythrin (PE)-conjugated anti-human -VEGFR-1, -VEGFR-2 and -VEGFR-3 (RD systems). (B) For double staining, HVMECs were fixed, permeabelised and double stained with either VEGFR3-PE/Prox-1-FITC (left panel) or VEGFR3-PE/Lyve-1-FITC (right panel) antibodies. Overall, these results clearly show that the majority of these cells have a lymphatic-like phenotype.
- Figure S3. PLGF does not induce significant proliferation of HMVEC (PDF, 10.9 KB) -
Cells were seeded in triplicate in gelatinized 96-well plates in medium containing 0.1% FCS and left unstimulated or stimulated with VEGF-A 10 ng/ml, VEGF-C 300 ng/ml and PLGF 100 ng/ml (RD). Results indicate that PLGF does not induce significant proliferation of HMVEC.
- Figure S4. Effect of NRP2 knock-down on VEGFR-2 induced phosphorylation by VEGF-A and VEGF-C (PDF, 11 KB) -
HMVEC transfected with control or NRP-2 siRNA (100 nM each). Twenty four hours after transfection cells were stimulated with VEGF-A (100ng/ml) or VEGF-C (300ng/ml). After stimulation, cells were washed and lysed as described in material and method. Then, lysates were used to quantify VEGFR-2 phosphorylation by ELISA. Results indicate that in this experiment VEGF-C induced a higher level of VEGFR-2 phosphorylation than VEGF-A. Blunting NRP2 reduced the level of VEGFR-2 phosphorylation induced by both stimuli. In conclusion, in both experiments, we showed that NRP-2 specific siRNA was able to reduce the level of VEGFR-2 phosphorylation in response to VEGF-A and VEGF-C. However, the level of VEGFR-2 phosphorylation and the extent of siRNA inhibition in response to these ligands may vary between experiments. This could be due to the state of these primary cells and also to the siRNA transfection efficiency, which may slightly vary between experiments.
- Figure S5. Short stability of FITC-siRNA after transfection in HMVEC (PDF, 36.6 KB) -
For the set up of HMVEC transfection with siRNA, cells were transfected with 100 nM of either FITC-conjugated siRNA (Invitrogen) or control siRNA (filled blue), using the Amaxa Nucleofector technology (Amaxa, GmbH). The fluorescence of transfected cells was monitored at different times by flow cytometric analysis. Results indicate that all cells were transfected with the siRNA. The level of siRNA in cells was acceptable 24h and 48h after transfection, but dramatically decreased thereafter.
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