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

Fig. 1. Generation and expression of a functional HaloTag-NRP1 construct in ECs.

Fig. 1

a HaloTag protein tag fused at the N-terminus of mouse NRP1 (HT-NRP1); sp, signal peptide; HT, HaloTag; a1/a2, complement C1r/C1s, Uegf, Bmp1 (CUB) domain 1/2; b1/b2, coagulation factor VIII/V (FVIII/FV) domain 1/2; c, meprin/A5/µ-phosphatase (MAM) domain. b Western blot analysis of exogenous HT-NRP1 construct and endogenous α-Tubulin in ECs transduced with empty pCCL lentivirus (Ctrl) or carrying HT-NRP1 construct. A representative experiment out of three is shown. c, d HaloTag NRP1 localizes at the cell membrane when endocytosis is inhibited, whereas it co-localizes with the EEA1 endocytic marker during internalization. Fluorescent confocal microscopy on ECs transduced with pCCL HT-NRP1, incubated for 15 min at 4 °C (to inhibit endocytosis) with the cell impermeable HaloTag-Alexa 660-labeled chloroalkane (magenta) fluorescent ligand (c), and then allowed to internalize membrane proteins by a shift to 37 °C (d). ECs were simultaneously stained for the endogenous early endosomal marker EEA1 (green). HaloTag-Alexa 660 bound HT-NRP1 localized on the surface of ECs kept at 4 °C (c) and is efficiently endocytosed only upon incubation at 37 °C for 3 min, where HT-NRP1 co-localized in EEA1-positive early endosomes (d yellow arrows). Bottom panels are magnifications of top panels. Scale bar is 10 µm. A representative experiment out of three is shown. e VEGF-A165 promotes the association of HT-NRP1 to VEGFR-2. Empty pCCL (Ctrl) or pCCL-HaloTag-NRP1 transduced ECs (HT-NRP1) were labeled with 3 µM HaloTag cell impermeable Biotin ligand for 15 min at 4 °C (to inhibit endocytosis) and stimulated or not with VEGF-A165 (30 ng/ml, 10 min at 37 °C), then immunoprecipitated on streptavidin-beads. A representative experiment out of two is shown. f HT-NRP1 rescues the defective adhesion of siNRP1 ECs to fibronectin. Results are the mean of three independent experiments (each in technical triplicate) ±SEM. Statistical analysis: parametric one-way analysis of variance (ANOVA) with Bonferroni correction; *P value = 0.0199, **P value = 0.0041. Source data are provided as Source Data file.