Mouse anti ptyrosine 4g10

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).

soluble Tie2-Fc chimera human (sTie2) (C-68028, Promokine, Germany, 5 µg/mL), D-Glucose (G7021, Sigma, Germany 5 mM or 30 mM), gelatin from porcine skin (48720, Fluka, Bucharest, Romania, 1% solution in PBS) was used to coat cell culture and assay plates. The antibodies used were: mouse anti-NDPK-B (Kamiya Biomedicals, Seattle, WA, USA; MC-412); mouse anti-Ang2 (sc-74403, Santa Cruz Biotechnology, Dallas, TX, USA); rabbit anti-Tie2 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-324); mouse anti-pTyrosine 4G10 (Millipore, Burlington, MA, USA; 05-321); mouse anti-γ-tubulin (Sigma-Aldrich, St. Louis, MO, USA; T6557); rabbit anti-Gβ (Santa Cruz Biotechnology, Dallas, TX, USA; sc-378); goat anti-Histone H3 (Santa Cruz Biotechnology, Dallas, TX, USA; sc-8654); Isolectin-TRITC (Sigma-Aldrich, St. Louis, MO, USA; L5264); goat anti-Tie2 (R&D Systems, Minneapolis, MN, USA; AF762); mouse anti-Tie2/TEK clone AB33 (Merck, Darmstadt, Germany; 05-584) rabbit anti-goat peroxidase (Sigma-Aldrich, St. Louis, MO, USA; A8919); rabbit anti-mouse peroxidase (Sigma-Aldrich, St. Louis, MO, USA; A9044); goat anti-rabbit peroxidase (Sigma-Aldrich, St. Louis, MO, USA; A9169); donkey anti-goat-FITC (Acris Antibodies, Hiddehausen, Germany; R1254F); swine anti-rabbit-FITC (Dako Agilent Technologies, Santa Clara, CA, USA; F0205)