Phosphorylated vegfr2

Standard western blotting of whole cell lysates was performed with antibodies for Stat3, Stat3 phosphorylated at Tyr705 (Cell Signaling Technologies), VEGF, VEGFR2, and phosphorylated VEGFR2 (Santa Cruz Biotechnology). β-actin (Sigma) was used as a control for loading.

Papilloma tissue samples were obtained for standard histological and immunohistological analyses prior to and following the first infusion of bevacizumab in two patients. For immunohistochemistry, formalin-fixed and paraffin-embedded tissues were analyzed using primary polyclonal rabbit anti-human antibodies specific to VEGF (Santa Cruz Biotechnology, CA, USA; working dilution, 1:50), phosphorylated VEGFR-2 (Santa Cruz Biotechnology; working dilution, 1:50) and the alkaline phosphatase anti-alkaline phosphatase double-bridge technique (DakoCytomation, Glostrup, Denmark). Briefly, tissue sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Following antigen retrieval in a microwave oven at 450 W (twice for 7 min in 10 mM sodium citrate [pH 6.0; DakoCytomation]) the primary antibodies were applied to the tissue sections overnight at 4°C. Subsequent steps were performed according to the manufacturer’s instructions. The Fast-Red substrate (DakoCytomation) was used for detection of phosphatase activity and sections were counterstained with hematoxylin and eosin (Merck KGaA, Darmstadt, Germany).