Tvegfr2

Serum starved PAE-KDR cells were incubated for 10 min with 1.5 nM VEGF (in all experiments VEGF-A₁₆₅ was used) with or without 30 min pretreatment with scFvs. Cells were lysed with lysis buffer (50 mM Tris (pH 7.5), 100 mM NaCl, 0.5% (w/v) Triton X-100) supplemented with protease inhibitor cocktail (Complete Mini EDTA-free, Roche) and phosphatase inhibitors (200 μM Na₃VO₄, 20 μM phenylarsine oxide). Lysates were diluted with 5× loading buffer (0.25 M Tris-HCl pH 6.8, 0.5 M DTT, 10% SDS, 50% glycerol, 0.5% bromophenol blue). Samples were boiled at 50 °C for 30 min, and resolved by 7% SDS PAGE, transferred to PVDF membranes (GE Healthcare, Piscataway, NJ, USA), and immunodecorated with primary antibodies (dilution 1:1000), followed by secondary alkaline phosphatase-coupled antibodies (1:10000). Immunoblots were developed with Novex AP Chemiluminescent Substrate (Invitrogen, Carlsbad, CA, USA). Amersham Imager 600 (GE) was used for analysis. Antibodies used were as follows: pVEGFR-2 (2478, Cell Signaling), tVEGFR-2 (2479, Cell Signaling), pPLCγ1 (2821, Cell Signaling), tPLCγ1 (2822, Cell Signaling), pAKT (4060, Cell Signaling), tAKT (4051, Cell Signaling), p38 (4631, Cell Signaling), and tp38 (9212, Cell Signaling). Protein marker used was PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (26619, ThermoFisher, Waltham, MA, USA).