Rabbit anti tie2

Heart tissue samples from anterior or basolateral left ventricular walls were obtained from explanted hearts after transplantation and were snap frozen within less than 4 h. Frozen samples were then oriented, and 6 µ-thick cryostat sections were cut and immunostained with a panel of primary antibodies applied in TRIS-buffered saline and with the use of appropriate secondary antibodies and fast-red substrate. The following panel of primary antibodies was used: goat anti angiogenin, Santa Cruz, sc-1408 (1:150); goat anti Ang-1, Santa Cruz, sc-6319 (1:200); goat anti Ang-2, Santa Cruz, sc-7016 (1:200); rabbit anti Tie-2, Santa Cruz, sc-9026 (1:100); goat anti procollagen-I, Santa Cruz, sc-8782 (1:50). Human tonsil or nasal polyps were used as a positive control. For the negative control slides, normal goat or rabbit nonspecific immunoglobulins (Santa Cruz Biotechnology) were used.

Cells seeded on 10 cm dishes were washed with ice-cold PBS and harvested by scraping in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100) containing cOmplete™ (protease inhibitor 1 tablet/10 mL buffer, Roche, Germany) and PhosStop™ (phosphatase inhibitor, 1 tablet/10 mL buffer Sigma Aldrich, Germany). The lysate was incubated on ice for 20–30 min and centrifuged at 13,000× g for 15 min at 4 °C. The supernatant was collected and incubated with the Tie2 precipitating antibody (AF313, R&D, UK, 2 µg per IP) on a top-down shaker at 4 °C for 2 h. 60 μL of the supernatant was set aside as Input control. For each sample, 40 μL of agarose beads were washed by lysis buffer. The beads were added to the lysate antibody mixture and incubated on a rotator at 4 °C overnight. After the incubation, the beads were collected by centrifugation and washed with lysis buffer containing PhosStop™. The immune complexes on the beads were then denatured with 1X Laemmli buffer, and the lysates were investigated by immunoblotting. Detection of Tie2 was performed with rabbit anti-Tie2 (Santa Cruz, Heidelberg, Germany, 1:500), and of phosphorylation with mouse-anti-pTyrosine 4G10 (05-321, Millipore, Darmstadt, Germany, 1:1000).