Journal: Nature Communications

Article Title: Endothelial Unc5B controls blood-brain barrier integrity

doi: 10.1038/s41467-022-28785-9

Figure Lengend Snippet: a Unc5B gene deletion strategy using tamoxifen injection in adult mice. b Immunofluorescence staining with the indicated antibodies and confocal imaging of adult brain sections at P67, 30 min after i.v cadaverine injection and reproduced on n = 4 Unc5B fl/fl and n = 5 Unc5BiECko brains. c Quantification of dye content in brains and peripheral organs at P67, 30 min after i.v. cadaverine injection, n = 11 Unc5B fl/fl and n = 10 Unc5BiECko brains (exact p -value = 0.0000397); n = 5 Unc5B fl/fl and n = 10 Unc5BiECko lungs, kidneys and hearts; n = 6 Unc5B fl/fl and n = 5 Unc5BiECko GI tracts. Each dot represents one mouse. d Immunofluorescence staining with the indicated antibodies and confocal imaging of adult brain sections, reproduced on n = 4 Unc5B fl/fl and n = 4 Unc5BiECko brains. e Quantification of endomucin+ vascular density. Each dot represents the mean of several images, n = 6 Unc5B fl/fl and n = 7 Unc5BiECko brains. One control mouse value was set as 1. f Tile-scan confocal imaging of adult Unc5BiECko brain sections at P67, 30 min after i.v cadaverine injection and reproduced on n = 3 Unc5B fl/fl and n = 3 Unc5BiECko brains. g Immunofluorescence staining of Unc5B and tile-scan confocal imaging of brain sections, reproduced on n = 4 mice. Boxes show higher magnifications of cortical areas. h Immunofluorescence staining with the indicated antibodies and confocal imaging of adult brain sections, reproduced on n = 4 Unc5B fl/fl and n = 4 Unc5BiECko brains. Arrowhead: Unc5B+/CD13+ pericyte. i , j Unc5B fl/fl was crossed with PDGFRβCre ERT2 and BBB permeability was assessed at P67, 30 min after i.v. cadaverine injection, n = 6 Unc5B fl/fl and n = 10 Unc5BiPCko brains. Each dot represents one mouse. All data are shown as mean ± SEM. NS non-significant, RSP Retrosplenial cortex, PTL Posterior parietal association areas, SSp Primary somatosensory cortex, PIR Piriform cortex, HI Hippocampus, HY Hypothalamus, TH Thalamus, ST Striatum, CB Cerebellum, M Medulla. Two-sided Mann–Whitney U test was performed for statistical analysis. Source data are provided as a Source Data file.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4 °C under gentle rotation with 10 ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Injection, Immunofluorescence, Staining, Imaging, Permeability, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endothelial Unc5B controls blood-brain barrier integrity

doi: 10.1038/s41467-022-28785-9

Figure Lengend Snippet: a Unc5B gene deletion strategy using tamoxifen injection in adult mice. Western blot ( b ) and quantification ( c ) of brain protein extracts at P67. Quantification of Caveolin-1 expression was performed on n = 4 Unc5B fl/fl and n = 4 Unc5BiECko brains. Quantification of ZO1, Occludin, GFAP and PDGFRβ expression was performed on n = 5 Unc5B fl/fl and n = 5 Unc5BiECko brains. Each dot represents one mouse. One control mouse was set as 1. d , e Immunofluorescence staining with the indicated markers and confocal imaging of brain sections, reproduced on n = 4 Unc5B fl/fl and n = 4 Unc5BiECko brains. Western blot ( f ) and quantification ( g ) of P67 brain protein extracts, n = 5 Unc5B fl/fl and n = 5 Unc5BiECko brains. Each dot represents one mouse. One control mouse value was set as 1. h Immunofluorescence staining with the indicated antibodies and confocal imaging of P67 piriform cortex 30 min after i.v cadaverine injection, reproduced on n = 4 Unc5B fl/fl and n = 4 Unc5BiECko brains. Arrowheads: larger vessels. All data are shown as mean ± SEM. NS non-significant. Two-sided Mann–Whitney U test was performed for statistical analysis. Source data are provided as a Source Data file.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4 °C under gentle rotation with 10 ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Injection, Western Blot, Expressing, Immunofluorescence, Staining, Imaging, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endothelial Unc5B controls blood-brain barrier integrity

doi: 10.1038/s41467-022-28785-9

Figure Lengend Snippet: a qPCR analysis of P67 brain mRNA extracts, n = 4 Unc5B fl/fl and n = 5 Unc5BiECko brains for quantification of LEF1 and PLVAP mRNA levels, n = 9 Unc5B fl/fl and n = 9 Unc5BiECko brains for quantification of CLDN5 mRNA levels. Each dot represents one mouse. One control mouse was set as 1. Western blot ( b ) and quantification ( c ) of Wnt/β-catenin signaling components in brain protein extracts, n = 7 Unc5B fl/fl and n = 8 Unc5BiECko brains. Each dot represents one mouse. One control mouse was set as 1. Immunofluorescence ( d ) and quantification ( e ) of LEF1 staining on adult brain sections. Each dot is the mean of several images, n = 6 Unc5B fl/fl and n = 6 Unc5BiECko brains. One control mouse was set as 1. f CTRL IgG and Unc5B immunoprecipitation on cultured brain endothelial cells, reproduced on n = 3 independent experiment. g Schematic of Unc5B adenoviral constructs. CTRL IgG or GFP immunoprecipitation in Unc5B siRNA knockdown ECs infected with siRNA resistant Unc5B adenovirus ( h ), and quantification of LRP6 pulldown ( i ), n = 4 independent experiment. Each dot represents one independent experiment. j Unc5B and Ctnnb1 gene deletion strategy using tamoxifen injection in adult mice. k Quantification of cadaverine content in P67 brains, 30 min after i.v. cadaverine injection, n = 4 Unc5B fl/wt , n = 3 Unc5B fl/wt iECko, n = 4 Ctnnb1 fl/wt , n = 4 Ctnnb1 fl/wt iECko, n = 6 Unc5B fl/wt ;Ctnnb1 fl/wt and n = 6 Unc5B fl/wt ;Ctnnb1 fl/wt iECko brains. Each dot represents one mouse. l Unc5B gene deletion and Ctnnb1 flex/3 gene overexpression strategy using tamoxifen injection. Western blot ( m ) and quantification ( n ) of P67 brain protein extracts, n = 4 Unc5BiECko and n = 5 Unc5BiECko;Ctnnb1 flex/3 brains. Each dot represents one mouse. One control mouse was set as 1. o Quantification of P67 brain cadaverine content, 30 min after i.v cadaverine injection, n = 5 Unc5BiECko and n = 5 Unc5BiECko;Ctnnb1 flex/3 brains. Each dot represents one mouse. All data are shown as mean ± SEM. NS non-significant. Two-sided Mann–Whitney U test was performed for statistical analysis between two groups. ANOVA followed by Bonferroni’s multiple comparisons test was performed for statistical analysis between multiple groups. Source data are provided as a Source Data file.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4 °C under gentle rotation with 10 ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Western Blot, Immunofluorescence, Staining, Immunoprecipitation, Cell Culture, Construct, Infection, Injection, Over Expression, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endothelial Unc5B controls blood-brain barrier integrity

doi: 10.1038/s41467-022-28785-9

Figure Lengend Snippet: a Unc5B deletion and eGFP::Claudin- 5 gene overexpression strategy using tamoxifen injection. b Quantification of P67 brain cadaverine content, 30 min after i.v cadaverine injection, n = 4 Unc5BiECko and n = 4 Unc5BiECko;eGFP::Claudin-5 brains. Each dot represents one mouse. Western blot ( c ) and quantification ( d ) of P67 brain protein extracts, n = 7 Unc5B fl/fl and n = 8 Unc5BiECko brains. Each dot represents one mouse. One control mouse was set as 1. e Unc5B and VEGFR2-Y949F gene recombination strategy using tamoxifen injection. f Quantification of P67 brain cadaverine content, 30 min after i.v cadaverine injection, n = 4 Unc5B fl/fl ;Y949F and n = 6 Unc5BiECko;Y949F brains. Each dot represents one mouse. Western blot ( g ) and quantification ( h ) of brain protein extracts, n = 7 Unc5B fl/fl and n = 8 Unc5BiECko brains. Each dot represents one mouse. One control mouse was set as 1. Immunofluorescence with the indicated antibodies ( i ) and quantification ( j ) of VE-cadherin coverage on P67 brain sections. Each dot is the mean of several images, n = 3 Unc5B fl/fl and n = 5 Unc5BiECko brains. One control mouse was set as 1. All data are shown as mean ± SEM. NS non-significant. Two-sided Mann–Whitney U test was performed for statistical analysis. Source data are provided as a Source Data file.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4 °C under gentle rotation with 10 ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Over Expression, Injection, Western Blot, Immunofluorescence, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endothelial Unc5B controls blood-brain barrier integrity

doi: 10.1038/s41467-022-28785-9

Figure Lengend Snippet: a , b Quantification of cadaverine content in P67 brains, 30 min after i.v. cadaverine injection. Ntn1 gene deletion was induced by tamoxifen injection between P60 and P64, n = 4 Ntn1 fl/fl , n = 4 Ntn1iko , n = 5 Robo4 +l+ and n = 4 Robo4 +l− brains. Each dot represents one mouse. Western blot ( c ) and quantification ( d ) of brain protein extracts, n = 7 Ntn1 fl/fl and n = 10 Ntn1iko brains. Each dot represents one mouse. One control mouse was set as 1. Western blot ( e ) and quantification ( f ) of mouse brain ECs treated with scrambled CTRL or Unc5B siRNA for 48 h and treated with recombinant mouse Netrin-1 (500 ng/ml) or not (−) for the indicated times. Each dot represents one independent experiment, n = 4 independent experiment. g CTRL IgG or Unc5B immunoprecipitation of brain protein extracts and Western blot for LRP6. h quantification of LRP6 pulldown with Unc5B, n = 3 Ntn1 fl/fl and n = 6 Ntn1iko brains. Each dot represents one mouse. One control mouse was set as 1. Western blot ( i ) and quantification ( j ) of mouse brain ECs treated with recombinant mouse Netrin-1 (500 ng/ml) or not (−) for the indicated times. Each dot represents one independent experiment, n = 4 independent experiment. Western blot ( k ) and quantification ( l ) of mouse brain ECs treated with CTRL DMSO or FAK inhibitor (5 uM) for 30 min followed by recombinant mouse Netrin-1 treatment (500 ng/ml) or not (−) for 1 h. Each dot represents one independent experiment, n = 3 independent experiment (pFAK-Y397(/β-actin): exact p -value between DMSO + Netrin1 (1 h) and FAKi + Netrin1 (1 h) = 0.000031). All data are shown as mean ± SEM. NS non-significant. Two-sided Mann–Whitney U test was performed for statistical analysis between two groups. ANOVA followed by Bonferroni’s multiple comparisons test was performed for statistical analysis between multiple groups. Source data are provided as a Source Data file.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4 °C under gentle rotation with 10 ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Injection, Western Blot, Recombinant, Immunoprecipitation, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endothelial Unc5B controls blood-brain barrier integrity

doi: 10.1038/s41467-022-28785-9

Figure Lengend Snippet: a , b Surface Plasmon Resonance measurements of anti-Unc5B-3 binding to human and rat Unc5B-ECD-Fc. c Dissociation constant for anti-Unc5B-3 binding to human and rat Unc5B. d Unc5B gene deletion strategy using tamoxifen injection. e Anti-Unc5B-3 was i.v. injected in P67 Unc5B fl/fl or Unc5BiECko mice for 15 min. Mice were perfused and anti-Unc5B-3 binding was detected by immunofluorescence on brain sections using an anti-human IgG antibody, reproduced on n = 4 Unc5B fl/fl and n = 3 Unc5BiECko brain. Western-blot ( f ) and quantification ( g ) of ECs treated with CTRL IgG or anti-Unc5B-3 for 1 h followed by recombinant mouse Netrin-1 treatment (500 ng/ml) for 10 min or 30 min. Each dot represents one independent experiment, n = 4 independent experiment. h Unc5B immunoprecipitation with a commercial antibody (R&D systems) of brain protein extracts from mice i.v injected with CTRL or anti-Unc5B-3 antibodies (1 h, 10 mg/kg), and western blot with antibodies recognizing the indicated ligands. i Quantification of h , n = 5 CTRL anti-Unc5B-1 and n = 5 anti-Unc5B-3 treated animals. Each dot represents one mouse. One control mouse was set as 1. j I.v. antibody injection strategy. k Immunofluorescence staining on brain sections from antibody-injected mice. l Quantification of brain cadaverine content, n = 5 CTRL IgG, n = 5 anti-Unc5B-3, n = 5 CTRL anti-Unc5B-1 and n = 4 anti-Unc5B-2 treated animals. Each dot represents one mouse. m I.v. antibody injection strategy. n Quantification of brain cadaverine content, n = 4 CTRL anti-Unc5B-1, n = 4 anti-Unc5B-3 and n = 3 anti-Unc5B-2 treated animals. Each dot represents one mouse. o I.v. antibody injection strategy. p , q Quantification of cadaverine content in peripheral organs, n = 4 CTRL anti-Unc5B-1, n = 5 anti-Unc5B-2, n = 5 CTRL IgG and n = 5 anti-Unc5B-3 treated animals. Each dot represents one mouse. All data are shown as mean ± SEM. NS non-significant. Two-sided Mann–Whitney U test was performed for statistical analysis between two groups. ANOVA followed by Bonferroni’s multiple comparisons test was performed for statistical analysis between multiple groups. Source data are provided as a Source Data file.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4 °C under gentle rotation with 10 ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: SPR Assay, Binding Assay, Injection, Immunofluorescence, Western Blot, Recombinant, Immunoprecipitation, Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endothelial Unc5B controls blood-brain barrier integrity

doi: 10.1038/s41467-022-28785-9

Figure Lengend Snippet: a Immunofluorescence staining of Human IgG, Claudin-5 and PLVAP and confocal imaging on brain sections from mice i.v. injected with anti-Unc5B-3 (10 mg/kg) for 1 h, 8 h or 24 h, reproduced on n = 3 untreated brains, n = 4 anti-Unc5B-3 treated brains for 1 h, n = 3 anti-Unc5B-3 treated brains for 8 and n = 3 anti-Unc5B-3 treated brains for 24 h. b CTRL IgG or Unc5B immunoprecipitation of brain protein extracts from mice i.v. injected with anti-Unc5B-3 and protein quantification ( c ), n = 3 control IgG treated brains, n = 4 anti-Unc5B-3 treated brains for 1 h and n = 3 anti-Unc5B-3 treated brains for 8 and n = 3 anti-Unc5B-3 treated brains for 24 h. Each dot represents one mouse. One control mouse was set as 1. All data are shown as mean ± SEM. NS non-significant. ANOVA followed by Bonferroni’s multiple comparisons test was performed for statistical analysis between multiple groups. Source data are provided as a Source Data file.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4 °C under gentle rotation with 10 ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Immunofluorescence, Staining, Imaging, Injection, Immunoprecipitation

Journal: Nature Communications

Article Title: Endothelial Unc5B controls blood-brain barrier integrity

doi: 10.1038/s41467-022-28785-9

Figure Lengend Snippet: Quantification of 10 kDa ( a ) 40 kDa ( b ) and 70 kDa ( c ) dextran content in P67 brains. Unc5B fl/fl and Unc5BiECko mice were injected with tamoxifen between P60-P64. CTRL anti-Unc5B-1, CTRL IgG, anti-Unc5B-2 and anti-Unc5B-3 antibodies were i.v. injected (10 mg/kg) for 1 h. Dextran was i.v. injected for 30 min. Quantification of 10 kDa dextran was performed on n = 5 Unc5B fl/fl and n = 5 Unc5BiECko brains, n = 4 CTRL anti-Unc5B-1, n = 5 anti-Unc5B-2, n = 5 CTRL IgG and n = 5 anti-Unc5B-3 treated brains. Quantification of 40 kDa dextran was performed on n = 11 Unc5B fl/fl and n = 10 Unc5BiECko brains (exact p -value = 0.000040), n = 4 CTRL anti-Unc5B-1, n = 4 anti-Unc5B-2, n = 5 CTRL IgG and n = 5 anti-Unc5B-3 treated brains. Quantification of 70 kDa dextran was performed on n = 5 Unc5B fl/fl and n = 5 Unc5BiECko brains, n = 5 CTRL IgG and n = 5 anti-Unc5B-3 treated brains. Each dot represents one mouse. d – f Quantification of 40 kDa dextran content in P67 organs, n = 5 Unc5B fl/fl and n = 5 Unc5BiECko brains, n = 4 CTRL anti-Unc5B-1, n = 4 anti-Unc5B-2, n = 5 CTRL IgG and n = 5 anti-Unc5B-3 treated brains. Each dot represents one mouse. g Quantification of P67 brain nanobody content 1 h after i.v CTRL IgG or anti-Unc5B-3 injection (10 mg/kg) and 30 min after i.v nanobody injection, n = 6 CTRL IgG and n = 7 anti-Unc5B-3 treated brains. Each dot represents one mouse. h Quantification of P67 brain and plasma BDNF concentration 1 h after i.v CTRL IgG or anti-Unc5B-3 injection (10 mg/kg) and 30 min in after i.v BDNF injection, n = 7 CTRL IgG and n = 7 anti-Unc5B-3 treated brains. Each dot represents one mouse. Trk-B immunoprecipitation of brain protein extracts from mice i.v. injected with CTRL IgG or anti-Unc5B-3 antibodies (10 mg/kg) for 1 h ( i ) and quantification of phospho-tyrosine pulldown ( j ), n = 5 CTRL IgG and n = 5 anti-Unc5B-3 treated brains. Each dot represents one mouse. One control mouse was set as 1. All data are shown as mean ± SEM. NS: non-significant. Two-sided Mann–Whitney U test was performed for statistical analysis between two groups. Source data are provided as a Source Data file.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4 °C under gentle rotation with 10 ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Injection, Concentration Assay, Immunoprecipitation, MANN-WHITNEY