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Blood, Vol. 114, Issue 15, 3343-3351, October 8, 2009
Phosphorylation of endothelial nitric oxide synthase by atypical PKC contributes to angiopoietin-1–dependent inhibition of VEGF-induced endothelial permeability in vitro
Blood Oubaha and Gratton
114: 3343
Supplemental materials for: Oubaha and Gratton
Files in this Data Supplement:
- Figure S1. Ang-1 stimulation counteracts VEGF-induced transendothelial permeability and NO production (JPG, 129 KB)
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(A) Transendothelial permeability was determined by measuring the passage of FITC-dextran through a monolayer of BLMVEC. Passage of FITC-dextran was measured following exposure of BLMVEC to VEGF, Ang-1, or VEGF and Ang-1 in combination (at the indicated concentrations). The data represent permeability to FITC-dextran expressed as the mean fold increases ± SEM with respect to untreated cells. (B) NO released in the culture media of BLMVEC subjected to 30 min stimulation with Ang-1 (100 ng/ml), VEGF (40 ng/ml) or both. Samples of culture media were taken for nitrite quantification as described under “Methods.”
- Figure S2. Tie2 activation by Ang-1 does not alter activation of VEGFR-2 by VEGF (JPG, 199 KB)
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BAEC were stimulated with VEGF (40 ng/ml), Ang-1(100 ng/ml) or both simultaneously for the indicated time. VEGFR-2 and Tie2 tyrosine phosphorylation levels were monitored in VEGFR-2 and Tie2 immunoprecipitates (IP). Equal immunoprecipitation levels from BAEC lysates were confirmed by western blotting (wb). These experiments were repeated at least 3 times with identical results.
- Figure S3. Overexpression of kinase-dead PKCζ inhibits Ang1-induced eNOS phosphorylation at Thr497 (JPG, 93.8 KB)
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BAEC were transfected with an empty vector or with an expression vector coding for kinase-dead PKCζ (KD-PKCζ). 48h post-transfection cells were starved for 6 hours before stimulation with Ang-1 (100ng/ml) for the indicated times. Total cells lysates were analyzed for p-Thr497-eNOS. Total eNOS and total PKCζ levels were monitored to confirm equal protein loading and overexpression, respectively.
- Figure S4. PKCζ knockdown in BLMVEC reverses the inhibition by Ang-1 of eNOS, NO release and of endothelial permeability (JPG, 336 KB)
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(A) BLMVEC were transfected with siRNA against PKCζ or control siRNA. 48h following transfections, cells were starved for 6 hours before Ang-1 stimulation (100ng/ml) for the indicated times. Membranes were probed with antibodies against p-Thr497-eNOS and eNOS. PKCζ knockdown was confirmed by western blotting (wb). (B) BLMVEC were transfected with PKCζ-siRNA (35uM; filled bars) or control siRNA (35uM; open bars). 48h post-transfection cells were starved for 6 hours before stimulation with VEGF (40ng/ml), Ang-1 (100ng/ml), or both for 30 min. Samples of culture media were taken for the quantification of NO released as described under “Methods.” PKCζ knockdown was confirmed by western blotting and eNOS protein levels were monitored to confirm equal protein loading (inset). (C) Permeability to FITC-dextran was determined in confluent BLMVEC monolayers transfected with PKCζ-siRNA (35uM; filled bars) or control siRNA (35uM; open bars) before stimulation with VEGF (40ng/ml), Ang-1 (100ng/ml), or both for 30 min. Data represent permeability to FITC-dextran expressed as the mean fold increase ± SEM with respect to untreated cells. PKCζ knockdown was confirmed by western blotting and eNOS protein levels were monitored to confirm equal protein loading (inset). * P<0.05.
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