Rabbit monoclonal anti vegfr 2

After 10 min of stimulation with recombinant human VEGF-A (10 ng/ml), recombinant human VEGF-C (400 ng/ml) (R&D Systems, Abingdon, UK) or MSC conditioned medium, cells were rinsed with ice-cold PBS and lysed with RIPA (Radio-Immunoprecipitation Assay) buffer containing phosphatase and protease inhibitors (Roche, Mannheim, Germany). VEGFR-3 phosphorylated proteins were isolated by binding to antibody directed against phosphotyrosine (1/100; mouse monoclonal anti-phosphotyrosine, Becton Dikinson, Franklin Lakes, NJ), overnight at 4°C. After 4 h precipitation using protein A sepharose beads (GE Healthcare, Diegem, Belgium), proteins were released from the beads by heating 5 min at 95°C in sample buffer (50 mM TrisHCl pH6.8, 4% SDS, 1% beta-mercaptoethanol, 20% glycerol, 0,05% bromophenol blue) and were subjected to VEGFR-2 and -3 Western-Blotting experiments (1/1000; rabbit monoclonal anti-VEGFR-2, Cell Signaling, San Diego, CA and 1/1000; mouse monoclonal anti-VEGFR-3, Millipore, Carrigtwohill, Ireland).

We performed western blotting analysis to validate the cross reactivity of the primary antibodies, using lysates from the MCT‐1 cell line developed in our laboratory from a grade III MCT derived from a 7‐year‐old male castrated Shar‐pei dog. The blots were blocked with 3% bovine serum albumin in TBS‐T (10 mmol/L Tris–HCl pH 7.5, 150 mmol/L NaCl, 0.1% Tween‐20) for 2 h and incubated overnight with the following primary antibodies: mouse monoclonal anti‐α‐tubulin (diluted 1:400 000; Sigma‐Aldrich, Oakville, ON, Canada), rabbit monoclonal anti‐VEGFR2 (1:2000), anti‐phospho‐VEGFR2 (1:2000) or anti‐NRP‐1 (1:1000), or rabbit polyclonal anti‐c‐CBL (1:1000); all from Cell Signaling Technology, Danvers, MA, USA. Membranes were washed three times for 5–10 min in TBS‐T and incubated in HRP‐labelled goat polyclonal anti‐mouse or anti‐rabbit secondary antibodies (both at 1:20 000; Sigma‐Aldrich) for 1 h. Membranes were incubated with chemiluminescent HRP substrate Luminata™ Forte (EMD Millipore, Darmstadt, Germany) and bands were visualized in a Bio‐Rad ChemiDocTM XRS+ system (Bio‐Rad Laboratories Canada Inc., Mississauga, ON, Canada).