Journal: The Journal of Cell Biology
Article Title: Polydom/SVEP1 binds to Tie1 and promotes migration of lymphatic endothelial cells
doi: 10.1083/jcb.202208047
Figure Lengend Snippet: Polydom does not induce Tie1 phosphorylation. (A) Serum-starved LECs were treated with EBM-MV2 medium containing 0.5% FBS, 1 μg/ml Polydom, or 500 ng/ml Ang1 at 37°C for 15 min. Immunoprecipitates (IP) of Tie1 (left) and Tie2 (right) from cell lysates were immunoblotted under reducing conditions for phosphotyrosine residues (upper panels) followed by reimmunoblotting for total Tie1 or Tie2 (lower panels). (B) 293-F cells were transfected with the indicated expression plasmids for Tie1 and Tie2 and treated with 1 μg/ml Polydom or 500 ng/ml Ang1 at 37°C for 15 min. Immunoprecipitates of Tie1 and Tie2 from cell lysates were immunoblotted under reducing conditions for phosphotyrosine residues (upper panels), followed by reimmunoblotting for total Tie1 or Tie2 (lower panels). Co-transfection of Tie2 with Tie1 increased Tie1 phosphorylation irrespective of the presence or absence of Polydom (lanes 4, 8, and 12). No Tie1 phosphorylation was induced by Polydom without Tie2 co-transfection (lanes 2, 6, and 10). Signals for phosphorylated Tie2 (pTie2; closed stars) were detected in Tie1 immunoprecipitates from Tie2-transfected cells (uppermost panel; lanes 3, 4, 7, 8, 11, and 12) because the anti-Tie1 polyclonal antibody used for the immunoprecipitation crossreacts with Tie2. Weak signals (open stars) were detected at (or slightly above) the position of pTie2 in the Tie1 immunoprecipitates from cells that were either untransfected or only transfected with Tie1 (lanes 1, 2, 5, 6, 9, and 10). Because Tie2 was not transfected in these cells, the weak signals (open stars) at the pTie2 position should be derived from tyrosine-phosphorylated proteins that were endogenously expressed in 293-F cells and nonspecifically precipitated with the anti-Tie1 antibody used. Such bands were not detected in untransfected or Tie1-transfected cells after immunoprecipitation with an anti-Tie2 antibody (lower panels; lanes 1, 2, 5, 6, 9, and 10). Source data are available for this figure: .
Article Snippet: The cells were starved in Endothelial Cell Basal Medium (EBM)-MV2 (PromoCell) overnight, detached, and resuspended in EBM-MV2 containing 0.1% BSA to a density of 5 × 10 5 cells/ml.
Techniques: Transfection, Expressing, Cotransfection, Immunoprecipitation, Derivative Assay
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