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J. Zhurinsky, M. Shtutman, and A. Ben-ze'ev, PAR3 (Millipore), and F-actin (Molecular Probes). Images were taken with a confocal microscope (Olympus FSV 1000), Visualization was obtained after digitized image multichannel deconvolution by AutoDeblur (Media Cybernetics Inc.), and three-dimensional projections were digitally reconstituted from stacks of confocal optical slices by Imaris software (Bitplane), vol.20, pp.4238-4252, 2000.

, Cell culture/DNA transfection and drug treatments HUVECs and HMVECs were cultured in EGM-2 or in EGM-2MV medium, respectively. HeLa cells were cultured in RPMI supplemented with 10% fetal bovine serum and penicillin-streptomycin. Primary endothelial cells from control (iEC WT) and Pdzrn3-deleted (iEC KO) mice were isolated from kidneys after magnetic separation with MACS MS columns (Miltenyi), p.20

, SiRNAs were transfected using interferin (Polyplus) at a final concentration of 30 nM. The oligonucleotides used were designed by Origene for h-Pdzrn3, pp.5-8

5. Sr307944b,

5. Sr307944c, 5?-GGAAUAUUUGUGUAGGUGAtt-3?; 08, 5?-AUUGCCUAAGGACUUGUGCUUtt-3?) as previously reported (47), and for hPKCz Stealth RNAi (#HSS18348) as previously reported (19efficacy of Pdzrn3, MUPP1, and PKCz knockdown was assessed by immunoblotting

, V5 (Invitrogen), and MUPP1 (Abcam) were used for immunoprecipitation assays. Proteins were then resolved by SDS-polyacrylamide gel electrophoresis and blotted with antibodies specific for V5 (Invitrogen), claudin 5 (Millipore), ZO1 (Invitrogen), PKCz (Santa Cruz Biotechnology), and phospho-PKCz (Ozyme Cell Signaling). Binding of antibodies to the blots was detected using the Odyssey Infrared Imaging System, p.3

, After a centrifugation at 800g for 5 min, the pellet was reserved for nuclear extract preparation, and the supernatant was collected and spun at 100,000g for 1 hour. The pellet and supernatant were classified as the "cell membrane fraction" and "cytoplasmic fraction," respectively. The pellet obtained after the centrifugation at 800g was washed and placed as a cushion on top of a 1 M sucrose buffer. After a centrifugation at 2700g for 5 min, the pellet was resuspended in a buffer containing tris, Cell fractionation For cell fractionation, cell or tissue lysates were resuspended in a buffer [containing Pipes (pH 6.8), 10 mM KCl, 1.5 mM MgCl 2 , 250 mM sucrose, 1 mM EGTA, and 1 mM EDTA] and potterized 20 times on ice

, Permeability assay HUVECs were seeded on inserts with a pore size of 0.4 mm (BD Biosciences), cultured in medium, and assayed for permeability to FITC dextran

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D. For-discussions, ;. Massey-harroche, and T. Miller, ) for providing the FLAG-tagged MUPP1 plasmid. Funding: This work was supported by grants from Fondation pour la Recherche Médicale (DVS20131228623) and from Region Aquitaine (20101301039MP). R.N.S. was supported by a grant from Region Aquitaine (20101301039MP) and from Fondation pour la Recherche Médicale (FDT20130928208). V.J. was supported by a grant from Fondation pour la Recherche Médicale (DEA20130726937), ) for her support with MUPP1 reagents, D. Vivien (GIP Cyceron, Caen, France) for discussions and his help in AVC training, and D. Daret (Inserm U1034

B. J. , -. , H. K. , and S. ,

V. J. , T. W. , and N. ,

M. P. , E. R. , and P. D. , were involved in study design; T.C. and C.D. designed the experiments, analyzed and interpreted the data, and edited the manuscript. All authors discussed the results and commented on the manuscript. Competing interests: The authors declare that they have no competing interests, 2016.

, , 2017.

R. N. Sewduth, H. Kovacic, B. Jaspard-vinassa, V. Jecko, T. Wavasseur et al., PDZRN3 destabilizes endothelial cell-cell junctions through a PKCz-containing polarity complex to increase vascular permeability, Sci. Signal, vol.10, p.3209, 2017.

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