Goat anti β3

Proteins were analyzed by SDS-PAGE and transferred to Immobilon P membranes. Blocking was performed by incubating the membranes with Tris-buffered saline (TBS) pH 7.4 with 0.05% Tween (TBS-T), containing either 5% nonfat dry milk or 3% BSA. Membranes were incubated with primary antibodies for 16 h at 4°C under continuous agitation, washed 3 times with TBS-T, and incubated with secondary antibodies for 1 h at room temperature. Primary antibodies used were mouse anti-Flk-1 (1:500), goat anti-β₃ (1:500), mouse anti-VEGF (1:500), rabbit anti-phospho-β₃(Y773) (1:1,000; Santa Cruz Biotechnology Inc.), mouse anti-RPTPβ/ζ (1:500; BD Biosciences), rabbit anti-c-Src (1:1,000; Merck Millipore), rabbit anti-phospho-c-Src(Y418) (1:1,000; Acris Antibodies GmbH) and rabbit anti-non-phospho-c-Src (1:1,000; Cell Signaling Technology Inc.). Detection of immunoreactive bands was performed using the enhanced chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL, USA). Protein levels were quantified using the ImagePC image analysis software (Scion Corp., Frederick, MD, USA).

Tumor tissues obtained after in vivo imaging were fixed in 4 % paraformaldehyde overnight. Specimens were then dehydrated in graded ethanol, embedded in paraffin, and sectioned at 5 μm on a Reichert microtome. The sections were blocked with 1 % BSA in PBS for 30 min and then incubated with goat anti-β₃ (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-VEGFR2 antibodies (1:100; Santa Cruz Biotechnology) overnight at 4 °C. Subsequently, sections were stained with FITC-labeled anti-goat and rhodamine-labeled anti-rabbit antibodies (1:200; Santa Cruz Biotechnology). Hoechst 33342 (1 μg/mL; Cell Signaling Technology, Danvers, MA, USA) was used to stain the cell nuclei of tumor tissues. Stained tissue sections were examined under a microscope (×200; Nikon Eclipse 80i).