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. 2015 Aug 28;24(22):6374–6389. doi: 10.1093/hmg/ddv349

Figure 3.

Figure 3.

Supplemental mutations accelerate TIE2 degradation at the plasma membrane. (A) HUVECs were transduced with bicistronic TIE2-IRES-GFP, left unstimulated or stimulated with Ang1 for 30 min and lysed. TIE2 and GFP were analyzed in western blot and signal intensities were quantified. In non-stimulated Y897F-R915L expressing cells, the total amount of TIE2 protein is reduced. Ang1-stimulation reduced TIE2 amount in TIE2-WT, but not in Y897F-R915L. **P < 0.01, n = 12, means + SD; NS, non-significant in two tailed t-test. (B) Plasma membrane located protein fractions were extracted in a biotinylation experiment, amounts of TIE2 and VE-Cadherin (as a control) were compared in western blot, and signal intensities were quantified. The plasma membrane located Y897F-R915L is less than in TIE2-WT (**P < 0.01 in two tailed t-test, n = 10). (C) HUVECs were transfected with bicistronic TIE2-IRES-GFP, TIE2 protein (left) and mRNA (middle) were quantified in western blot and quantitative PCR and protein/mRNA ratio was calculated (right). Y897F-R915L protein (*P < 0.05 versus TIE2-WT in ANOVA followed by Tukey's post hoc test) but not mRNA is reduced. Single mutations show a trend for additive effect for diminished protein/mRNA ratio. (D) Protein synthesis was inhibited using cycloheximide and total protein was collected in the indicated time-points, and measured in TIE2 ELISA. In R915L and Y897F-R915L, the TIE2 protein amount decreased faster than in TIE2-WT. *P < 0.05 and **P < 0.01 in ANOVA followed by Tukey's post hoc test; means + SD, n = 2–4 independent experiments run in duplicates. (E) Amount of soluble TIE2 in the conditioned medium was measured in TIE2 ELISA from HUVECs expressing TIE2 forms indicated and normalized to TIE2 amount in corresponding cell lysates and total protein concentration. Amount of shed TIE2 increases in Y897F-R915L and in R915L when compared with TIE2-WT (TIE2-WT set to 1.0, ***P < 0.001, **P < 0.01 versus TIE2-WT and #P < 0.05 versus Y897F in ANOVA followed by the Tukey's post hoc test, n = 4).