Expression and activation of Tie2 receptors in HUVECs and neutrophils. Tie2 mRNA expression in neutrophil (N) was revealed by RT-PCR, HUVEC (EC) mRNA was used as a positive control, and base pairs DNA ladder (L) was used as the molecular-weight standard. In contrast, Tie1 was present in HUVECs but not in neutrophils (A). Tie2 expression in neutrophils was confirmed at the protein level by immunocytochemistry, using anti-hTie2 IgG, and normal IgG was used as negative control (B). Tie2 protein was located at the cytoplasmic membrane of neutrophils as demonstrated by confocal microscopy (C). HUVECs were treated with either Ang1, Ang2, or Ang1 and Ang2 (10^–9 M; 7.5 minutes). Tie2 proteins were first immunoprecipitated (IP) with anti-hTie2 IgG and Tie2 phosphorylation was analyzed by immunoblotting using antiphosphotyrosine IgG (4G10 clone). WB indicates Western blot. Membranes were subsequently stripped and the detection of Tie2 protein was performed to confirm equal protein loading (D). Neutrophils were treated with Ang1, Ang2, or Ang1 and Ang2 (10^–9 M; 7.5 minutes). Cell lysates equivalent to 125 μg total proteins were loaded in each lane. Membranes were subsequently probed with antiphospho–hTie2 IgG (E).