RGM-C/Hemojuvelin Antibody

Detection of RGM-C/Hemojuvelin by Immunohistochemistry

Soluble HFE2 and RGMa effects on endothelial cells.a Knockout of liver Hfe2 reduces pericyte coverage in section from the cerebral cortex (mean ± s.e.m.; unpaired two-tail t-test; AAV8-GFP n = 3, Hfe2 delta Alb-cren = 4, and AAV8-AlbCre n = 3). Scale bar, 50μm. b Quantification through quantitative RT-PCR shows a reduction in PDGF-B mRNA levels in liver knock out animals (mean ± s.e.m.; paired two-tail t-test; Hfe2fl/fln = 3 and Hfe2 delta Alb-cren = 3). c Quantification of PDGF-B mRNA in cultured bEnd.3 cells following indicated treatments. Treatment with RGMa leads to a significant reduction of PDGF-B mRNA levels that is rescued with HFE2 addition (mean ± s.e.m.; paired two-tail t-test; HFE2 n = 3, RGMa n = 4, and RGMa+ HFE2 n = 5; n representing an independent experiment). d Immunocytochemistry of claudin-5 in human primary endothelial cell monolayer after PBS, HFE2, RGMa, and RGMa+HFE2 treatments and quantification (mean ± s.e.m.; unpaired two-tail t-test; replicates n = 3). Scale bar, 100 µm. e Western blotting of Claudin-5 expression in bEnd3 cell lysates after indicated treatment (mean ± s.e.m.; unpaired two-tailed t-test; replicates n = 3). f Transwell permeability leakage assay performed on a monolayer of bEnd3 cells using HRP (mean ± s.e.m.; paired one-tail t-test; replicates n = 3). g Transwell permeability leakage assay performed on a monolayer of human primary endothelial cells using 70 kDa FITC-dextran (mean ± s.e.m.; paired two-tail t-test; replicates n = 3). h In vitro TEER analysis shows that RGMa alters the integrity of a monolayer of bEnd3 cells, this is rescued by HFE2 (mean ± s.e.m.; unpaired two-tail t-test; replicates n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file (includes exact p-values). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38310100), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of RGM-C/Hemojuvelin by Immunohistochemistry

Hfe2-deficient mice displays severe BCB disruption.a Schematic of the BCB assessment method using light-sheet microscopy. b 3D rendered representative light-sheet microscopic images of TR-dextran deposition in CUBIC-cleared brains (scale bars, 400 µm) and time-series quantification (mean ± s.e.m.; unpaired two-tail t-test; Hfe2fl/fl (control) n = 5, Hfe2 delta AlbCren = 6, AAV8-GFP n = 5, AAV8-AlbCre n = 7; n representing data generated from one brain). c Representative in-vivo multiphoton images of TR-dextran at 40 mins time-point (scale bar, 50 µm) from the parietal lobes of the cerebral cortex. The normalized extravascular fluorescence ( delta F/F) intensity was plotted over time (mean ± s.e.m.; unpaired two-tail t-tests) and the groups differences were assessed with spatial leakage log-likelihood (repeated measures factorial ANOVA rendered as approximate unpaired two-tail t-test; Hfe2fl/fln = 5, Hfe2 delta AlbCren = 10, AAV8-GFP n = 6, AAV8-AlbCre n = 5). d Representative confocal images of fibrinogen disposition around the vessels of cerebral cortex (isolectin) of Hfe2-deficient mice (scale bar, 50 µm) and quantification (mean ± s.e.m.; unpaired two-tail t-test; Hfe2fl/fln = 4, Hfe2 delta AlbCren = 5, AAV8-GFP n = 5, AAV8-AlbCre n = 6). e Confocal imaging of TR-dextran deposition in brain slices from the cerebral cortex with endothelium counterstain. The same observation was made in sections from 3 AAV8-GFP and 3 AAV8-AlbCre brains. f 3D rendered representative light-sheet microscopic images of TR-dextran deposition in CUBIC-cleared brains (scale bars, 400 µm; mean ± s.e.m.; unpaired two-tail t-test; Hfe2fl/fln = 5, Hfe2 delta ActaCren = 6). *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file (includes exact p-values). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38310100), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of RGM-C/Hemojuvelin by Immunohistochemistry

Hfe2-deficient mice displays severe BCB disruption.a Schematic of the BCB assessment method using light-sheet microscopy. b 3D rendered representative light-sheet microscopic images of TR-dextran deposition in CUBIC-cleared brains (scale bars, 400 µm) and time-series quantification (mean ± s.e.m.; unpaired two-tail t-test; Hfe2fl/fl (control) n = 5, Hfe2 delta AlbCren = 6, AAV8-GFP n = 5, AAV8-AlbCre n = 7; n representing data generated from one brain). c Representative in-vivo multiphoton images of TR-dextran at 40 mins time-point (scale bar, 50 µm) from the parietal lobes of the cerebral cortex. The normalized extravascular fluorescence ( delta F/F) intensity was plotted over time (mean ± s.e.m.; unpaired two-tail t-tests) and the groups differences were assessed with spatial leakage log-likelihood (repeated measures factorial ANOVA rendered as approximate unpaired two-tail t-test; Hfe2fl/fln = 5, Hfe2 delta AlbCren = 10, AAV8-GFP n = 6, AAV8-AlbCre n = 5). d Representative confocal images of fibrinogen disposition around the vessels of cerebral cortex (isolectin) of Hfe2-deficient mice (scale bar, 50 µm) and quantification (mean ± s.e.m.; unpaired two-tail t-test; Hfe2fl/fln = 4, Hfe2 delta AlbCren = 5, AAV8-GFP n = 5, AAV8-AlbCre n = 6). e Confocal imaging of TR-dextran deposition in brain slices from the cerebral cortex with endothelium counterstain. The same observation was made in sections from 3 AAV8-GFP and 3 AAV8-AlbCre brains. f 3D rendered representative light-sheet microscopic images of TR-dextran deposition in CUBIC-cleared brains (scale bars, 400 µm; mean ± s.e.m.; unpaired two-tail t-test; Hfe2fl/fln = 5, Hfe2 delta ActaCren = 6). *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file (includes exact p-values). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38310100), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of RGM-C/Hemojuvelin by Western Blot

Soluble HFE2 and RGMa effects on endothelial cells.a Knockout of liver Hfe2 reduces pericyte coverage in section from the cerebral cortex (mean ± s.e.m.; unpaired two-tail t-test; AAV8-GFP n = 3, Hfe2 delta Alb-cren = 4, and AAV8-AlbCre n = 3). Scale bar, 50μm. b Quantification through quantitative RT-PCR shows a reduction in PDGF-B mRNA levels in liver knock out animals (mean ± s.e.m.; paired two-tail t-test; Hfe2fl/fln = 3 and Hfe2 delta Alb-cren = 3). c Quantification of PDGF-B mRNA in cultured bEnd.3 cells following indicated treatments. Treatment with RGMa leads to a significant reduction of PDGF-B mRNA levels that is rescued with HFE2 addition (mean ± s.e.m.; paired two-tail t-test; HFE2 n = 3, RGMa n = 4, and RGMa+ HFE2 n = 5; n representing an independent experiment). d Immunocytochemistry of claudin-5 in human primary endothelial cell monolayer after PBS, HFE2, RGMa, and RGMa+HFE2 treatments and quantification (mean ± s.e.m.; unpaired two-tail t-test; replicates n = 3). Scale bar, 100 µm. e Western blotting of Claudin-5 expression in bEnd3 cell lysates after indicated treatment (mean ± s.e.m.; unpaired two-tailed t-test; replicates n = 3). f Transwell permeability leakage assay performed on a monolayer of bEnd3 cells using HRP (mean ± s.e.m.; paired one-tail t-test; replicates n = 3). g Transwell permeability leakage assay performed on a monolayer of human primary endothelial cells using 70 kDa FITC-dextran (mean ± s.e.m.; paired two-tail t-test; replicates n = 3). h In vitro TEER analysis shows that RGMa alters the integrity of a monolayer of bEnd3 cells, this is rescued by HFE2 (mean ± s.e.m.; unpaired two-tail t-test; replicates n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file (includes exact p-values). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38310100), licensed under a CC-BY license. Not internally tested by R&D Systems.