Supplemental material and methods

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1 Supplemental material and methods Antibodies: Primary antibodies used for these stainings were 174/2 1 (migg1 against PV-1), PAL-E 2 (migg2a; Abcam, Cambridge, UK), anti-nrp-1 (monoclonal migg2a or polyclonal sheep IgG; R&D Systems, Minneapolis, USA), 2C8 3,4 (migg1, against CD31/PECAM-1), anti-cd31 (clone M-20, polyclonal goat IgG, Santa Cruz Biotechnology, California, USA), or negative class-matched control antibodies AK-1 (migg1; In Vivo Biotech Services, Hennigsdorf, Germany), migg2a (R&D Systems), normal sheep serum.(abd Serotec, Oxford, UK) and normal goat serum (Vector laboratories, California, USA). Tissues: Normal human liver, choroid plexus, thymus and heart were collected during autopsies. Peripheral lymph nodes and tonsils were collected during routine surgery. The use of human material and all procedures were approved by the Ethical Board of Turku University Hospital and abided by the Declaration of Helsinki. Immunofluorescence: 6 µm thick sections were cut on a Leica CM3050S cryostat (Leica Microsystems, Wetzlar, Germany), fixed for 3 minutes in ice-cold acetone and stored at -70ºC until used. Sections were stained for 30 min. with primary antibodies diluted in PBS, and incubated for 30 min. with isotype specific secondary antibodies fluorescently labeled with Alexa fluor dyes. Secondary antibodies were diluted in PBS, 10% human AB-serum. Where indicated, free secondary antibody binding sites were blocked with 5% species specific serum in PBS for 10 min. Sections were then incubated with second primary antibodies followed by isotype specific Alexa fluor labeled secondary antibody as before. After every step

2 sections were washed for 2x5 min. in PBS. Finally, sections were mounted in ProLong Gold mounting medium (Invitrogen, Carlsbad, USA) and stored at 4ºC until imaged. Stainings were analyzed on a LSM 510 confocal microscope (Carl Zeiss Microimaging, Göttingen, Germany). All images were acquired using the same settings using a Plan Neofluar 20x/0,5 air objective and LSM software (Carl Zeiss Microimaging). Only the detector gain was adjusted to obtain images with even fluorescence intensities. Stainings were analyzed at room temperature. Z-stacks for supplemental videos were acquired using a Plan Neofluar 40x/0,75 air objective with 0,5 µm spacing between single sections. Images and videos were processed using ZEN 2011 software (Carl Zeiss Microimaging). Antibody combinations: Primary Antibody migg2a neg. Secondary Antibody Blocking ) Primary Antibody AK-1 (migg1 Secondary Antibody contr. IgG2a Alexa 488 neg. contr.) IgG1 Alexa 546 PAL-E (migg2a) IgG2a Alexa /2 (migg1 anti PV-1) IgG1 Alexa 546 PAL-E (migg2a) IgG2a Alexa 488 2C8 (migg1 anti CD31) IgG1 Alexa 546 anti-nrp-1 (migg2a) IgG2a Alexa /2 (migg1 anti PV-1) IgG1 Alexa 546 anti-nrp-1 (migg2a) IgG2a Alexa 488 2C8 (migg1 anti CD31) IgG1 Alexa 546 goat serum donkey anti goat IgG Alexa 546 goat serum AK-1 (migg1 neg. contr.) IgG1 Alexa 488 anti-cd31 (goat IgG) donkey anti goat IgG Alexa 546 goat serum 174/2 (migg1 anti PV-1) IgG1 Alexa 488 sheep serum donkey anti sheep IgG Alexa 546 goat serum migg2a neg. contr. IgG2a Alexa 488 anti-nrp-1 (sheep IgG) donkey anti sheep IgG Alexa 546 goat serum PAL-E (migg2a) IgG2a Alexa 488

3 Flow cytometry: HEK EBNA cells were transiently transfected (Lipofectamine 2000, Invitrogen) with pcdna3.1 plasmids containing either PV-1, NRP-1 or empty vector following manufacturer's instructions. Cells were subsequently grown for hours, detached using trypsin EDTA and spun down. Stainings were performed for 30 min with primary antibodies diluted in PBS, 0,01% sodium azide at room temperature. After 3 washes in the same solution, cells were incubated with species specific FITC-labelled secondary antibodies diluted in PBS, 2% fetal bovine serum, 10% AB-serum, 0,01% sodium azide on ice. After 3 washes in PBS, 0,01% sodium azide, cells were resuspended in the same solution and analyzed on a FACS Calibur using CellQuest Pro software (both BD Biosciences, California, USA). Subsequent data analysis and preparation of figures were performed using Flowing Software (Perttu Terho, Centre for Biotechnology, Turku, Finland, ) Co-immunoprecipitations and immunoblotting: For western blot analyses, lymphocyte depleted tonsil lysate and HEK-PV-1 lysate were run on 10% SDS-PAGE under non reducing conditions and transferred to nitrocellulose membranes (GE Healthcare, Wisconsin, USA). After blocking of non-specific binding with 5% non-fat milk in phosphatebuffered saline, 0,1% Tween-20 for 1 hour, the membranes were probed with primary antibodies diluted in the same solution for 1 hour. The washed membranes were incubated with horseradish peroxidase conjugated secondary antibodies (rabbit anti-mouse IgG and rabbit anti-sheep IgG; both from Dako, Denmark) for 1 hour and subsequently visualized using ECL Western Blotting Detection Reagents (GE Healthcare). Co-immunoprecipitations were performed as described previously 5. Briefly, magnetic beads (Dynal, New York USA) were coated either with 174/2,

4 α-nrp-1 (polyclonal goat IgG; Santa Cruz), AK-1 (migg1 negative control antibody) or normal goat serum (Vector laboratories, California, USA). Fresh, lymphocyte depleted tonsil lysate or transfected HEK-EBNA lysates (either mock transfected or PV-1 and NRP-1 cotransfected as described for flow cytometry) were incubated with coated magnetic beads on a rotor at 4ºC. The beads were subsequently washed according to the protocol, snap frozen in liquid nitrogen and run in a speed-vac over night. Proteins were then resuspended in Laemmli sample buffer containing 5% mercaptoathanol. Samples were subsequently analyzed as described above. Polyclonal goat anti-nrp-1 antibodies were detected using horseradish peroxidase coupled donkey anti-goat IgG secondary antibody (Santa Cruz Biotechnology). 1. Niemela H, Elima K, Henttinen T, Irjala H, Salmi M, Jalkanen S. Molecular identification of PAL-E, a widely used endothelial-cell marker. Blood. 2005;106: Schlingemann RO, Dingjan GM, Emeis JJ, Blok J, Warnaar SO, Ruiter DJ. Monoclonal antibody PAL-E specific for endothelium. Lab Invest. 1985;52: Henttinen T, Jalkanen S, Yegutkin ) GG. Adherent leukocytes prevent adenosine formation and impair endothelial barrier function by Ecto-5'- nucleotidase/cd73-dependent mechanism. J Biol Chem. 2003;278: Airas L, Salmi M, Jalkanen S. Lymphocyte-vascular adhesion protein-2 is a novel 70-kDa molecule involved in lymphocyte adhesion to vascular endothelium. J Immunol. 1993;151: Cristea IM, Williams R, Chait BT, Rout MP. Fluorescent proteins as proteomic probes. Mol Cell Proteomics. 2005;4:

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8 Supplemental Figure Legend: Supplemental Figure 1. Double stainings of PAL-E, PV-1 and NRP-1 with the prototype vessel marker CD31. Double stainings of PAL-E, PV-1 and NRP-1 with CD31 were performed as described in materials and methods and supplemental materials and methods. CD31 stains vessels of blood vascular and lymphatic origin. White arrows mark structures, where the stainings are significantly different between the two antibodies. Scale bars represent 100 µm. Supplemental Figure 2. Negative controls for double stainings of PV-1, PAL-E and NRP-1. Appropriate negative control antibodies were used to perform control stainings to ensure specificity of the stainings in figure 1. Staining procedure and antibody combinations can be found in detail in supplemental materials and methods. Scale bars represent 100 µm. Supplemental Figure 3. Negative controls for double stainings of PV-1, PAL-E, NRP-1 and CD31. Appropriate negative control antibodies were used to perform control stainings to ensure specificity of stainings in figure 1. Staining procedure and antibody combinations can be found in detail in supplemental materials and methods. Scale bars represent 100 µm.

9 Supplemental video legend: Video 1: 3-dimensional reconstruction of a vessel stained with an anti-pv-1 antibody and PAL-E. A vessel of a thymus section stained with PAL-E (green) and anti-pv-1 antibodies (red) was imaged using a Plan Neofluar 40x/0,75 objective on a Zeiss LSM 510 confocal microscope. Images were acquired in 0,5 µm intervals and 3D reconstruction was performed using ZEN 2011 software. Direction of rotation and scale bar are provided in the movie. Video 2: 3-dimensional reconstruction of a vessel stained with anti-pv-1 and anti-nrp-1 antibodies. A vessel of a tonsil section stained with anti-nrp-1 (green) and anti-pv-1 (red) antibodies was imaged using a Plan Neofluar 40x/0,75 objective on a Zeiss LSM 510 confocal microscope. Images were acquired in 0,5 µm intervals and 3D reconstruction was performed using ZEN 2011 software. Direction of rotation and scale bar are provided in the movie. ) Video 3: 3-dimensional reconstruction of a vessel stained with an anti-nrp-1 antibody and PAL-E. A vessel of a liver section stained with PAL-E (green) and anti-nrp-1 antibody (red) was imaged using a Plan Neofluar 40x/0,75 objective on a Zeiss LSM 510 confocal microscope. Images were acquired in 0,5 µm intervals and 3D reconstruction was performed using ZEN 2011 software. Direction of rotation and scale bar are provided in the movie. Video 4: 3-dimensional reconstruction of a vessel stained with anti-pv-1 and anti-cd31 antibodies. A vessel of a tonsil section stained with anti-pv-1 (green) and anti-cd31 (red) antibodies was imaged using a Plan Neofluar 40x/0,75 objective on a Zeiss LSM 510 confocal microscope. Images were acquired in 0,5 µm intervals and 3D reconstruction was performed using ZEN 2011 software. Direction of rotation and scale bar are provided in the movie. Video 5: 3-dimensional reconstruction of a vessel stained with an anti-cd31 antibody and PAL-E. A vessel of a peripheral lymph node section stained with PAL-E (green) and anti-cd31 antibody (red) was imaged using a Plan Neofluar

10 40x/0,75 objective on a Zeiss LSM 510 confocal microscope. Images were acquired in 0,5 µm intervals and 3D reconstruction was performed using ZEN 2011 software. Direction of rotation and scale bar are provided in the movie. Video 6: 3-dimensional reconstruction of a vessel stained with anti-nrp-1 and anti-cd31 antibodies. A vessel of a liver section stained with anti-nrp-1 (green) and anti-cd31 (red) antibodies was imaged using a Plan Neofluar 40x/0,75 objective on a Zeiss LSM 510 confocal microscope. Images were acquired in 0,5 µm intervals and 3D reconstruction was performed using ZEN 2011 software. Direction of rotation and scale bar are provided in the movie. )