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. Author manuscript; available in PMC: 2021 Nov 8.
Published in final edited form as: Circ Res. 2020 Mar 16;126(10):1379–1393. doi: 10.1161/CIRCRESAHA.119.315730

Figure 6. LZTR1-mediated VEGFR2 internalization affects blood vessel integrity.

Figure 6.

(a, b) Lztr1fl/fl ECs were infected with an empty vector (EV) or Cre-recombinase (Cre) and treated with DMSO, Dynasore (40μM), or Tyrphostin A23 (10μM) for 12 hours. After permeabilization, the cells were immunostained with anti-VEGFR2 antibody. Scale bar 20 μm. Data shown as mean ± SEM; p-values were assessed by Wilcoxon Mann-Whitney test. (c) Levels of internalized VEGFR2 were assessed by immunostaining of Lztr1+/+ and Lztr1+/− ECs transfected with Scrambled siRNA or siRNA targeting Chmp1b. Data are presented as mean ± SEM; p-values were assessed by Wilcoxon Mann-Whitney test. (d) ELISA analysis of soluble VEGFR2 in the plasma of Lztr1+/+ and Lztr1 +/− mice. Data are present as mean ± SEM; p-values were assessed by Wilcoxon Mann-Whitney test. (e, f) Immunoblotting analysis of phosphorylated and total VEGFR2 in Lztr1fl/fl ECs infected with an empty vector (EV) or Cre-recombinase (Cre) and treated with DMSO, MLN4924 (1μM), or Tyrphostin A23(10μM) for 12 hours. Data shown as mean ± SEM; p-values were assessed by Mann-Whitney test. (g, h) Lztr1 fl/fl ECs transduced with empty vector (EV) or Cre-recombinase (Cre), treated for 12 hours with DMSO or cediranib (10μM), and immunostained with antibodies against ZO1 and VE-cadherin. Scale bar 20 μm. Quantification of the number of cells with junctional gaps is shown as mean ± SEM; p-values were assessed by Wilcoxon Mann-Whitney test to compare efficiency of each specific treatment within each group. (i) Evans blue extravasation from PdgfbiCreERT2, Lztr1 fl/fl and Lztr1 fl/fl mice skin after treatment with PBS or VEGF. Miles assay performed on the ears. N=4 mice per group. Data are normalized to tissue weight and shown as mean ± SEM; p-values were assessed by Wilcoxon Mann-Whitney test to compare efficiency of treatment within group. (j) Evans blue extravasation from Pdgf iCreERT2, Lztr1fl/fl and Lztr fl/fl mice skin after vehicle or cediranib treatment. Miles assay. N=4 mice per group. Data are normalized to tissue weight and shown as mean ± SEM; p-values were assessed by Wilcoxon-Mann-Whitney test to compare efficiency of treatment within group.