Anti nrp2

The following primary anti-human antibodies were used: sheep anti-podoplanin, goat anti- VEGFR-3, anti-LYVE-1, anti-PROX1, anti-ITGA9, anti-VEGFR-2, anti-NRP2, anti-podocalyxin and anti-GFP, all from R&D Systems, Minneapolis, MN. Mouse anti-human CD14 and anti-CD68 antibodies were from Santa Cruz Biotechnology, Dallas, TX and Thermo Fisher, Waltham, MA, respectively. Rabbit anti-acetylated histone H3 and anti-Ki-67 were from Upstate, Billerica, MA and Cell Signaling Technologies, Danvers, MA, respectively. Rabbit anti-mouse Lyve-1 and rat anti-Meca-32 antibodies were from AngioBio, Del Mar, CA, and BioXCell, West Lebanon, NH, respectively. Secondary antibodies conjugated to FITC, Cy3, DyLight 488, DyLight 549, and APC donkey anti-rabbit, anti-sheep, anti-rat, anti-mouse and anti-goat IgG were all from Jackson ImmunoResearch Laboratories (West Grove, PA).

Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in JS buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl, 5 mM EGTA pH 7.8, 10% glycerol, 1% Triton, 1.5 mM MgCl2, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF). The protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). For Western blot analysis, proteins were separated on SDS–PAGE, gels were blotted onto Immobilon P (Millipore, Bredford, MA, USA) for 2 h and the membranes were blocked in 5% nonfat dry milk in Tris–buffered saline for 2 h or overnight before the addition of the antibody for 1 h. The primary antibodies used were: anti-GAPDH (Santa Cruz, CA), anti-PAX8 (kindly provided by R. Di Lauro) anti-Tubulin (Santa Cruz, CA) and anti-NRP2 (R&D SYSTEMS). The filters were washed three times in Tris–buffered saline plus 0.05% Tween 20 before the addition of horseradish peroxidase-conjugated secondary antibodies for 45 min. Horseradish peroxidase was detected with ECL (Pierce).