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. 2022 Jul 20;13:4188. doi: 10.1038/s41467-022-31904-1

Fig. 5. Mini-WARS is an extracellular inhibitory NRP1 ligand that impairs VE-cadherin turnover.

Fig. 5

a Schematic showing the different domains in human Full Length (FL, red), mini (green) and T2 (blue) WARS isoforms; WHEP, vertebrate-specific extension; ESE, eukaryote-specific extension. b Endogenous NRP1 and mini-WARS co-immunoprecipitation. An anti-WARS Ab recognizing the catalytic Rossmann fold and the anticodon recognition domain detected only the mini-WARS band (green arrow) co-immunoprecipitating with NRP1. A representative experiment out of five is shown. c Pull-down experiment identifies mini-WARS as a preferential binding partner of NRP1-Fc. A representative western blot from three independent experiments is shown. d Mini-WARS interaction with NRP1 does not rely on its cytoplasmic domain. A HA antibody used to detect WT or ∆cyto or ∆b1/b2 NRP1-HA co-immunoprecipitated with FL or T2 or mini-WARS-V5 co-transfected in ECs. The image is representative of three independent experiments. e S100A10 silencing increases the secretion of V5-mini-WARS in the EC extracellular medium. Control (siCtrl) or S100A10 (siS100A10) silenced ECs were transfected with mini-WARS V5 tagged and their medium loaded on V5 beads. A representative western blot from three independent experiments is shown. f Mini-WARS fosters NRP1 and VE-cadherin localization at endothelial cell-to-cell contacts. Confocal microscopy on pCCL empty lentivirus (Ctrl) or pCCL mini-WARS transduced ECs stained for NRP1 (magenta) and VE-cadherin (green); quantification of NRP1 (above) and VE-cadherin (below) fluorescence intensity in AJs. Data are the mean ± SEM of three independent experiments (depicted in different color shades). Scale bar is 10 µm. Statistical analysis: two-tailed heteroscedastic Student’s t test; *P value = 0.0164, ***P value = 0.0005. g Mini-WARS overexpression does not affect NRP1 and VE-cadherin protein levels in ECs. A representative experiment out of three is shown. h Mini-WARS overexpression decreases NRP1 and VE-cadherin internalization in ECs. Percentage of NRP1 (left) and VE-cadherin (right) endocytosis in Ctrl or mini-WARS-transduced ECs as measured by surface labeling followed by capture ELISA. Data are the mean ± SEM of three independent experiments. Statistical analysis: two-tailed heteroscedastic Student’s t test; *P value = 0.0201 (left), *P value = 0.0498 (right). Source data are provided as a Source Data file.