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Fig. 5. Ang1 and Ang2 induce differential internalization of Tie2 in HUVECs. Surface biotinylation of HUVECs was used to assess the amount of Tie2 remaining at the cell surface upon Ang1 and Ang2 stimulation. (A) Surface biotinylated HUVECs were lysed and incubated with streptavidin conjugated to agarose beads. Non-biotinylated cells (Non), biotinylated (Biotin), biotinylated followed by stripping (Biotin + Stripping) and total cell lysate (Cell Lysate) proteins were resolved by SDS-PAGE and immunoblotted using a monoclonal anti-Tie2 antibody. (B) HUVECs were incubated with pre-clustered Ang1 (800 ng/ml), or Ang2 (800 ng/ml) for the indicated times at 37°C. Cell surface proteins were biotinylated and isolated with streptavidin agarose followed by immunoblotting as described in (A), t=0 represents cells stimulated with media alone. (C) The intensities of the bands in B were quantified and plotted against time, Ang1 (
) Ang2 (
). Results are mean ± s.e.m. of four independent experiments.
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