P tie2

Tissue or cells were lysed in 2x Laemmli buffer and boiled for 5 min at 100 °C followed by centrifugation at 20,000 × g for 15 min at 4 °C. The protein extracts were subjected to standard Western blot analysis which was performed. The following antibodies were used for western blotting: Rabbit polyclonal antibody against CCM3 was generated (Invitrogen) against full-length recombinant human CCM3 protein expressed and purified from Escherichia coli. β-actin (mouse, A1978) and β-actin (mouse, A5441) were from Sigma; p-Caveolin-1 (rabbit, 611339) was from BD Pharmingen; Abl (rabbit, 2862s), p-Akt (rabbit, 9271), Akt (rabbit, 9272), p-MLC2 (rabbit, 3674), PDGFR-β (rabbit, 3168), p-Tie2 (rabbit, 4221), p-Tie2 (rabbit, 4226), Tie2 (rabbit, 4224), and VEGFR2 (rabbit, 2479) were from Cell Signaling Technology. Caveolin-1 (rabbit, sc894) was from Santa Cruz. Angpt2 (rabbit, ab8452), p-Abl (rabbit, ab4717), and N-Cadherin (rabbit, ab76057) were from Abcam; Angpt2 (AF7186) was from R&D Systems. All primary antibodies were diluted 1:1000. For data presented in the same figure panel, the samples were derived from the same experiment and that gels/blots were processed in parallel. Uncropped blots and gel images were provided in the Source data file.

TIE2-WT HUVECs were seeded on coverslips in a 24-well plate overnight. The cells were stimulated with angiopoietins (500 ng/ml) in M200 basal medium for 1 hr at 37°C in 5% CO₂. Cells were fixed in 4% PFA for 15 min, permeabilized in 0.5% TritonX-100 in PBS for 3 min and stained for anti-TIE2 (total TIE2, Ab33, Merck Millipore) and anti-phosphorylated TIE2 (pTIE2, Y992, Cell Signaling) antibodies. The cells were imaged with LSM780 confocal microscope (Carl Zeiss) with a Plan-Apochromat 40x/1.4 objective, and ratio of pTIE2/total TIE2 staining intensity at cell–cell junctions was quantified by using ZEN 2012 (blue edition) software (Carl Zeiss).