Journal: The FASEB Journal

Article Title: Endothelial neuropilin‐2 influences angiogenesis by regulating actin pattern development and α5‐integrin‐ p ‐FAK complex recruitment to assembling adhesion sites

doi: 10.1096/fj.202100286r

Figure Lengend Snippet: FIGURE 4 Long term depletion of NRP2 accelerates FA maturation and fibrillogenesis. A, Mean length of α5-integrin adhesions (µm), error bars show mean ± SEM; n ≥ 150 adhesions/timepoint/condition); Asterisks indicate statistical significance from an unpaired two-tailed t test. B, siRNA-transfected ECs were seeded onto 6 cm dishes pre-coated with 10 µg/mL FN and incubated for either 90, 180 min or 16 h at 37°C. ECs were lysed in ESB and subjected to the DC protein assay before being analyzed by western blotting using primary antibodies against Tensin-1. GAPDH was used as a loading control. C, Bands were quantified using ImageJ densitometric analysis. Error bar shows mean ± SEM; N = 3 independent experiments; asterisks indicate statistical significance from an unpaired two-tailed t test. D, siRNA-transfected ECs were seeded onto FN coated coverslips and allowed to adhere for 16 h at 37°C and 5% CO2 before being fixed. ECs were co-immunostained for α5-integrin and tensin-1. Images were taken using a Zeiss LSM880 Airyscan Confocal microscope at 63X magnification. XZ plane images for each panel are also shown to display colocalization. E, As described in D), ECs were allowed to adhere for either 16 for 48 h, before being co-immunolabeled with α5- integrin and EDA-FN. F, siRNA-treated ECs were allowed to adhere for 16 h at 37°C and 5% CO2. ECs were then lysed, cleared and the insoluble fraction isolated. Insoluble fractions were separated by SDS-PAGE and subjected to Western blot analysis. Membranes were incubated in anti- EDA-FN primary antibody to assess quantities of insoluble cell secreted FN, anti-NRP2 primary antibody to confirm NRP2 depletion, and anti- HSC70 antibody as a loading control. G, % insoluble EDA-FN. Mean densitometric analysis obtained using ImageJ. Error bars show mean ± SEM; N ≥ 3 independent experiments; asterisks indicate statistical significance from an unpaired two-tailed test. H/I, Quantification of EDA-FN density at either 16 h (H) or 48 h (I) adhesion to FN. Error bars show mean ± SEM; n ≥ 20 ECs/group. Asterisks indicate statistical significance from an unpaired two-tailed t test

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 4 of 19 | BENWELL Et aL. dilution and purchased from Cell Signalling Technology, unless noted otherwise) were: anti- NRP2 (clone D39A5), anti- HSC70 (clone B- 6; Santa Cruz Biotechnology), anti- pVASPSer157 (clone 3111), anti- VASP (clone 3132), anti- pFAKTyr407 (clone 44- 650G; Invitrogen), anti- tensin- 1 (clone NBP1- 84129; Novus Biologicals), anti- GAPDH (60004- 1- 1g; Proteintech), EDA- FN (F6140; Sigma).

Techniques: Two Tailed Test, Transfection, Incubation, DC Protein Assay, Western Blot, Control, Microscopy, Immunolabeling, Isolation, SDS Page