B10.2) (all: Santa Cruz), rabbit polyclonal α-plexA1 or -A4 (Abcam), mouse α-VSV-G (Sigma), mouse α-MV H protein (K83, produced in our laboratory) and mouse α-NP-1 (clone AD5-17F6, Miltenyi). For double stainings with mouse monoclonal antibodies, α-NP-1 was directly conjugated according to the manufacturer’s protocol (Zenon, Molecular Probes/Invitrogen). After final washing steps in PBS, fluorochrome G (Southern Biotech, Eching, Germany) was used as the mounting medium and cells

were analyzed by confocal laser scanning microscopy (Laser Scan Microscope, LSM510 Meta, Software version 3.0; Axiovert 200 microscope, objective: 100×; NA=1.4 Plan Apochromat). T cells were nucleofected with 2 μg plasmid encoding for DN-plexA1 (kindly provided by L. Tamagnone, Milano) 54 following the manufacturer’s protocol (Amaxa). For silencing of plexA1, human T cells were transfected with a two-day interval according ABT 263 to the manufacturer’s protocol (DharmaFECT, Thermal Scientific) with 100 nM siRNA targeting KU-60019 concentration plexA1 (Santa Cruz) or, for control, a scrambled siRNA (Sigma). Before cells were recruited into

the respective experiments, aliquots were harvested for nucleic acid extraction (Qiagen, RNAeasy Kit) and subsequent RT-PCR analyses. Forward 5′-ctgctggtcatcgtggctgtgct and reverse 5′-gggcccttctccatctgctgcttga primers were used for specific amplification of plexA1. Signals obtained Cell Penetrating Peptide after electrophoresis were digitalized and quantified using the AIDA software program (Raytest, Straubenhardt, Germany). Supernatants of DC or DC/T-cell co-cultures were harvested at the time intervals indicated and immunoprecipitated using 2 μg/mL rabbit polyclonal anti-SEMA3A antibody (H300, Santa Cruz). Immune complexes were washed in PBS containing 0.5 M LiCl and 1% v/v Triton X100, and analyzed by Western blot using an anti-SEMA3A mAb (R&D Systems) followed by an anti-mouse HRP-conjugated antibody (Dianova, Hamburg, Germany). Signals obtained after ECL development

were digitalized and quantified (recombinant SEMA3A-Fc was included for reference) using the AIDA software program. For conjugate analyses, DC were labelled with 1 μM R18 dye for 20 min and T cells with 1 μM CSFE (both: Invitrogen) for 5 min (each in RPMI-5% FBS at 37°C). DC and T cells (exposed to SEMA3A/6A or human IgG (150 ng/mL each) for 15 min at 37°C) were co-cultured directly in an FACS tube for the time intervals indicated, fixed with PFA (final concentration of 2% w/v in PBS), washed once with FACS buffer (low-speed centrifugation (400 rpm)) and subsequently analyzed by flow cytometry. The double-positive population representing conjugates was determined, and percentages were calculated using one sample t-test with hypothetical value set as 100 for the IgG-treated controls. Under-agarose assays were performed as described elsewhere 41. Briefly, 2.