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. 2021 Sep 28;220(11):e202006033. doi: 10.1083/jcb.202006033

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© 2021 Camillo et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

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Figure S2. — Endogenous FLRT2 expression, silencing, and LPHN2-mediated signaling impacts on endothelial cell adhesion and migration. (A) Real-time quantitative PCR analysis of FLRT1-3 mRNAs in human ECs relative to the housekeeping gene GAPDH. Results are the mean ± SD of four independent experiments. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; ***, P ≤ 0.001. (B) Real-time quantitative PCR analysis of LPHN2 and FLRT1-3 mRNAs in siCTL and siLPHN2 human ECs. Results are normalized on siCTL values and are the mean ± SD of four independent experiments. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; NS, P > 0.05; ***, P ≤ 0.001. (C) COS-7 cells transfected with empty vector or WT HA-Lphn2-EGFP or ΔOLF HA-Lphn2-EGFP constructs and their expression verified by Western blot (WB) analysis using a rat mAb anti-HA (middle). Next, COS-7 cells transfected with empty vector or WT HA-Lphn2-EGFP or ΔOLF HA-Lphn2-EGFP constructs were incubated or not with 6xHis-tagged rhFLRT2. The binding between the Lphn2 constructs and the rhFLRT2 ligand was revealed through sequential incubation with a mouse mAb anti-6xHis, an AP-conjugated goat anti-mouse pAb, and the AP substrate nitro blue tetrazolium–5-bromo-4-chloro-3-indolyl-phosphate (right). Results show how WT but not ΔOLF Lphn2 binds rhFLRT2. (D–F) Confocal microscopy analysis (D) of paxillin (Pax; blue), and phalloidin-labeled F-actin (red). Cells were first transduced with pCCL lentivirus (carrying GFP)-mediated overexpression (green) of silencing-resistant mouse WT or ΔOLF Lphn2 and then oligofected with either siCTL or siLPHN2 siRNAs. Scale bar, 10 µm. Confocal microscopy analysis reveals how lentiviral delivery of WT Lphn2, but not ΔOLF Lphn2 mutant, restores the phenotype of paxillin-containing FAs both considering the number (E) and the size (expressed by maximum Feret diameter, F). Results are the mean ± SD of two independent experiments for a total of 15 ECs for each condition. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; NS, P > 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (G) Real-time quantitative PCR analysis of FLRT2 mRNA in siCTL or siFLRT2 human ECs relative to the housekeeping gene GAPDH and normalized on siCTL levels. Results are the mean ± SD of six independent assays. Statistical analysis: two-tailed heteroscedastic Student’s t test; ***, P ≤ 0.001. (H) Western blot analysis with an anti-FLRT2 Ab of lysates of siCTL or siFLRT2 ECs or siFLRT2 ECs treated with exogenous rhFLRT2 (800 ng/ml). Endogenous FLRT2 appears as an ∼85 kD protein, while the soluble extracellular portion of exogenous rhFLTR2 appears as an ∼75 kD protein band. (I) LPHN2 silencing in human ECs decreased SHANK2 interaction with Rap1 small GTPase. Western blot analysis of Rap1 coimmunoprecipitated (IPed) with SHANK2 in cultured ECs. Results are the mean ± SD of three independent experiments. Statistical analysis: two-tailed heteroscedastic Student’s t test; *, P ≤ 0.05. Max., maximum; n., number.