Figure 3.

NRP1 Localizes into the Mitochondria and Interacts with ABCB8

(A) PLA for ABCB8 and phosphoserine in HDMECs si-control or si-NRP1; PLA with IgG isotypes was performed as control; scale bar, 20 μm.

(B) PLA signal (gray) per cells (mean ± SEM) was measured in a minimum of 177 cells from two independent experiments; # = number of cells analyzed.

(C) Quantification of ABCB8 mRNA by RT-qPCR in HDMECs si-NRP1 or si-control; n = 3.

(D) HDEMC si-NRP1 and si-control were stained for ABCB8 (green) and counterstained with DAPI (blue); scale bars, 20 μm.

(E) Integrated density of ABCB8 signal was quantified and expressed as percentage relative to si-control (mean ± SEM; n = 3).

(F) Aortas of Nrp1^fl/fl (Nrp1^WT) or Nrp1^fl/fl;Cdh5(PAC)-iCre^ERT2 (Nrp1^ECKO) littermates injected daily with tamoxifen for 5 days and sacrificed after 1 month from injections were immunostained for NRP1, ABCB8, and PECAM; scale bar, 30 μm.

(G) NRP1 and ABCB8 pixel number and intensity were measured in optical z stacks, normalized to DAPI, and expressed as percentage of Nrp1^WT (mean ± SEM; n ≥ 3 mice each genotype; **p < 0.005, ***p < 0.001, two-way ANOVA).

(H) Co-immunoprecipitation of endogenous NRP1 and ABCB8 protein from lysates of HDMECs performed with an anti-NRP1 antibody, followed by immunoblotting for NRP1 and ABCB8 (n = 3).

(I) PLA for NRP1 and ABCB8 in HDMECs si-control or si-NRP1. PLA with IgG isotypes was performed as control; scale bar, 20μm.

(J) Average PLA signal (gray) per cells (mean ± SEM) was measured in a minimum of 59 cells from four independent experiments.

# = number of cells analyzed; *p < 0.05; **p < 0.005, ***p < 0.001; n.s., not significant; Student's t test. See also Figures S1 and S2.