Human pro ngf

Surface Plasmon Resonance was performed on a Biacore T200 (GE Healthcare, Pittsburgh, PA) to measure binding affinities of each antibody to nerve growth factor (NGF). A 2.5 μg/ml cNGF in 10 mM Sodium Acetate pH 4 (GE Healthcare, BR-1003–49), 5 μg/ml human NGF (R&D Systems Cat #256-GF/CF) in 10 mM Sodium Acetate pH 4 and 1 μg/ml human proNGF (Alomone labs Cat. # N-280) in 10 mM Sodium Acetate pH 5 (GE Healthcare, BR-1003–51) was immobilized by amine coupling using EDC/ NHS for a final density ~250 RU (resonance unit) on CM5 sensor flow cells 2–4, respectively. Flow cell 1 is used as an internal reference to correct running buffer effects. Antibody binding was measured at 15°C with a contact time of 250 s and flow rate of 30 μl/min. The dissociation period was 300 s. Regeneration was performed with regeneration buffers (10 mM Glycine pH1.5 and 10 mM NaOH) and flow rate at 20 μl/min for 60 s each. Running/dilution buffer (1X HBS-EP, GE Healthcare, BR-1006–69, 10X including 100 mM HEPES, 150 mM NaCl, 30 mM EDTA and 0.5% v/v surfactant P20, pH7.4, 1:10 in filtered MQ H2O) was used as negative control at the same assay format. Data were analyzed with Biacore T200 Evaluation software by using the method of double referencing. The resulting curve was fitted with the 1:1 binding model.

Detailed information about cell lines is included in Supplementary Table 6. All melanoma cell lines were grown in high glucose DMEM (Lonza) supplemented with 10% fetal bovine serum (Hyclone), 2 mM glutamine and 20 μg/mL gentamicin. Primary melanocytes were cultured in 254CF medium (Gibco) supplemented with human melanocyte growth supplement (Gibco); melan-a⁶¹ were cultured in RPMI (Gibco), supplemented with 5% fetal calf serum (Gibco) and 200 nM 12-o-tetradecanoyl phorbol-13-acetate (Sigma). Human dermal lymphatic microvascular endothelial cells HMVEC-dLyAd and human lymph node endothelial cells HLEC were cultivated in Clonetics EGM-2 MV BulletKit (Lonza) following the manufacturer’s instructions. Human lymphatic fibroblasts HLF were cultured in high glucose DMEM (Lonza) supplemented with 10% fetal bovine serum (Hyclone). All cells were grown at 37°C in a humidified 5% CO2 atmosphere.
When indicated, HLECs were treated with the following compounds: NGFR inhibitor THX-B (15 μM) and MEKi PD0325901 (1 μm) synthetized by CNIO Experimental Therapeutics Program; NF-kB inhibitor JSH-23 (5 μM, Merck), GRGDSP peptide (10 ng/mL, Merck), anti-integrin α_v blocking antibody (0.1 μg/mL, Merck), Fc soluble ICAM-1 (10 μg/mL, R&D Systems), diphtheria toxin (10 μg/Kg mouse body weight, Sigma Aldrich), human Pro-NGF (50ng/mL, Alomone) and human BDNF (5 ng/mL, Peprotech).