Goat anti vegfr3

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).

Collected cells were lysed in SDS buffer and sonicated on ice. After quantifying protein concentrations using BCA reagent (Thermo Fisher Scientific, Waltham, MA, USA), proteins were resolved by SDS-PAGE and transferred to PVDF membrane by electroblot analysis. Membranes were blocked in Tris-buffered saline containing 5% skim milk and 0.05% Tween-20 and incubated with the following primary antibodies: goat anti-VEGFR3 (1:1000) (AF349) (R&D systems) and rabbit anti-β actin (1:8000) (Medical and Biological Laboratories, Nagoya, Aichi, Japan). Horseradish peroxidase conjugated anti-goat, anti-rabbit IgG (1:4000) were used as secondary antibodies for chemiluminescence detection. Signals were obtained by enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA). The bands were analyzed by densitometry using ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA).