Journal: Kidney international

Article Title: The L1 cell adhesion molecule is a potential biomarker of human distal nephron injury in acute tubular necrosis.

doi: 10.1038/sj.ki.5002640

Figure Lengend Snippet: Figure 4 | Comparative localization of L1 intra- and extracytoplasmic domains. (a) L1 scheme showing the extracellular part with six immunoglobulin-like domains (Ig-like) (including one recognized by mAb272) and five fibronectin III-like (FNIII) domains followed by the transmembrane segment and cytoplasmic fragments (recognized by the goat polyclonal serum). (b–d) Normal CDs are compared with (e–g) damaged CDs. (b, e) CDs are stained by goat polyclonal antiserum labeled by FITC (green) and (c, f) mAb272 labeled by rhodamine (red). (d, g) Merge. (d) In normal CD, both antibodies give a basolateral signal. (g) In injured principal cells, the basolateral signal is low with both antibodies, whereas there is a strong apical membrane (arrow) signal with the anti-cytoplasmic domain antibody contrasting with no signal for the extracellular fragment. Original magnification 630.

Article Snippet: After four washes with TBS-T (Tris-buffered saline with 0.1% Tween), goat anti-human L1 extracellular domain antibody was added (R&D Systems), followed by anti-goat coupled to alkaline phosphatase (Sigma-Aldrich, St Louis, MO, USA).

Techniques: Staining, Labeling, Membrane

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Nrg1β Released in Remote Ischemic Preconditioning Improves Myocardial Perfusion and Decreases Ischemia/Reperfusion Injury via ErbB2-Mediated Rescue of Endothelial Nitric Oxide Synthase and Abrogation of Trx2 Autophagy

doi: 10.1161/atvbaha.121.315957

Figure Lengend Snippet: Figure 1. Superoxide-induced Nrg1 (neuregulin 1) release by the microvascular endothelial bed of the hindlimb acts as remote ischemic preconditioning (RIPC)-factor and protects the myocardium from ischemia/reperfusion (I/R)-induced injury. A, Mice were either subjected to sham or three cycles of 5 min hindlimb ischemia followed by 5 min reperfusion. Thirty minutes after the last I/R cycle, serum Nrg1 levels were determined by ELISA. Mean±SEM was plotted as a bar graph (n=5). *P<0.01 vs sham. B, Formaldehyde fixed paraffine embedded gastrocnemius muscle sections from sham or RIPC-treated mice were immunostained with anti-Nrg1 (Continued )

Article Snippet: Recombinant human Nrg1β1 active domain (catalog No. 396-HB) and neutralizing anti-Nrg1 antibody (catalog No. AF-396-NA) were bought from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Nrg1β Released in Remote Ischemic Preconditioning Improves Myocardial Perfusion and Decreases Ischemia/Reperfusion Injury via ErbB2-Mediated Rescue of Endothelial Nitric Oxide Synthase and Abrogation of Trx2 Autophagy

doi: 10.1161/atvbaha.121.315957

Figure Lengend Snippet: Figure 2. Remote ischemic preconditioning (RIPC) improved cardiac perfusion via Nrg1 (neuregulin 1)-dependent eNOS (endothelial nitric oxide synthase) activation. A, Mice were subjected to sham or myocardial ischemia/reperfusion (I/R) or RIPC+I/R surgery, and at the end of the reperfusion period, the nontargeted contrast agent was injected to mice via femoral vein while collecting B-mode/contrast mode images of the heart from long-axis view using Vevo 3100. To block the function of circulating Nrg1 in mice, neutralizing anti-Nrg1 antibodies (150 µg/kg) was administered to mice before RIPC and then subjected to RIPC+I/R injury. B, Contrast intensities in the left ventricle (LV) anterior wall downstream to the ligation site were quantitated and plotted over time. Loss of peak height and the rightward shift of peak indicates hypoperfusion. C, From the rate of contrast agent intensity change, the perfusion index was calculated. Percentage of perfusion was calculated based on the perfusion index assuming its level in the sham group is 100% and blotted as a bar graph (n=5) and shown as a heat map over the anterior LV in A. Values are means±SEM (n=5 mice). *P<0.01 vs sham, **P<0.01 vs I/R, §P<0.01 vs RIPC+I/R (ANOVA). D, Left coronary artery from mice were isolated, and after indicated treatments, NO formation was detected by electron spin resonance (EPR) spectrometry using NO spin trap Fe2+-(N-methyl-D-glucamine dithiocarbamate) 2 (Fe-[MGD]2) as described in methods. E, NO-Fe(MGD) 2 spin count was quantified and plotted as a bar graph (n=5). *P<0.01 vs sham; **P<0.01 vs I/R (ANOVA). F–H, HMVECs were exposed to 2 h hypoxia followed by 1-hour reoxygenation, and the medium was collected (preconditioned medium [PM]). HCAECs were treated with PM for the indicated period, lysed, and cell lysates were collected. An equal amount of cell lysates were analyzed for eNOS activity (F) and plotted as a bar graph (n=3). *P<0.01 vs control (ANOVA). An equal amount of cell lysates were analyzed for eNOS phosphorylation at Ser 1177 by Western blotting using its phospho-specific antibodies and normalized with total eNOS levels (G), or the cell lysates were immunoprecipitated with anti-eNOS antibodies, and the immunoprecipitates were analyzed by Western blotting using anti-phospho-tyrosine specific antibodies (H). (Continued )

Article Snippet: Recombinant human Nrg1β1 active domain (catalog No. 396-HB) and neutralizing anti-Nrg1 antibody (catalog No. AF-396-NA) were bought from R&D Systems.

Techniques: Activation Assay, Injection, Blocking Assay, Ligation, Isolation, Electron Paramagnetic Resonance, Activity Assay, Control, Phospho-proteomics, Western Blot, Immunoprecipitation

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Nrg1β Released in Remote Ischemic Preconditioning Improves Myocardial Perfusion and Decreases Ischemia/Reperfusion Injury via ErbB2-Mediated Rescue of Endothelial Nitric Oxide Synthase and Abrogation of Trx2 Autophagy

doi: 10.1161/atvbaha.121.315957

Figure Lengend Snippet: Figure 3. Only preischemic and not postischemic Nrg1β (neuregulin 1) administration protects myocardium due to loss of endothelial-ErbB2 (Erb-B2 receptor tyrosine kinase 2) during ischemia. A, To determine the protective function of Nrg1 during ischemia or reperfusion, recombinant Nrg1β (4 µg/kg) was injected into mice before ischemia (preischemia) or after ischemia (postischemia) but before reperfusion. After ischemia/reperfusion (I/R), the heart was isolated and perfused with annexin-V-Fe complex. The infarcted tissue was excised, and tissue bound annexin-V was quantified by electron spin resonance (EPR). B, The absolute spin count was calculated from the EPR spectra of paramagnetic-Fe bound to annexin-V and plotted as a bar graph. Values are means±SD (n=5 mice). *P<0.01 vs sham; **P<0.01 vs I/R, †P<0.01 vs Nrg1 (preischemic) plus IR (ANOVA). C, Mice were subjected to sham, I/R, remote ischemic preconditioning (RIPC) or RIPC+I/R, and the infarcted tissue region was excised. Protein extract was prepared from the excised tissue and analyzed by Western blotting for ErbB2 and ErbB4 levels. D, Sections of sham and I/R mouse heart were subjected to immunofluorescence staining using anti-ErbB2 and anti-α-actinin (cardiomyocyte marker) antibodies. Isolectin-Alexa Fluor 647 conjugate was used to stain endothelial cells selectively. Fluorescent images of the stained sections were obtained using an upright Zeiss fluorescence microscope (AxioImager Z2) via 40×/1.4 NA objective. Scale bar=20 mm. E and F, Adult mouse cardiomyocytes and mouse cardiac endothelial cells were isolated from adult mouse heart, and cell lysate was prepared. An equal amount of protein from cell lysates was analyzed for ErbB2 levels by Western blotting. Blot was reprobed for α-actinin and CD31 (F). ErbB2 levels were quantified and plotted as a bar graph (n=3). *P<0.01 vs MCEC (Student t test). G, HCAECs were pretreated with preconditioned medium (PM) or Nrg1-neutralized PM, then exposed to hypoxia/reoxygenation (H/R), and cell lysates were prepared. An equal amount of protein from each sample was analyzed for ErbB2 and ErbB4 levels by Western blotting. H, HCAECs were pretreated with or without PM and exposed to H/R. At the end of treatment, cells undergoing apoptosis were labeled with annexin V-FITC conjugate, and the percentage of apoptotic cells was quantitated by fluorescence- activated cell sorting analysis using Attune NxT Flow Cytometer and plotted as a bar graph (n=3). *P<0.01 vs normoxia, **P<0.01 vs H/R (ANOVA). I, HCAECs were transfected with nontarget (NT) or ErbB4 or ErbB4 siRNA (100 nmol/L), and after recovery from transfection, they were treated with or without PM and exposed to H/R. Cells undergoing apoptosis were labeled with annexin V-FITC conjugate, and the percentage of apoptotic cells was quantitated and plotted as a bar graph (n=3). *P<0.01 vs NT siRNA normoxia, **P<0.01 vs NT siRNA H/R; †P<0.01 vs NT siRNA PM+H/R (ANOVA). ANOVA followed by Tukey post test was performed using GraphPad-Prism software (version 8).

Article Snippet: Recombinant human Nrg1β1 active domain (catalog No. 396-HB) and neutralizing anti-Nrg1 antibody (catalog No. AF-396-NA) were bought from R&D Systems.

Techniques: Recombinant, Injection, Isolation, Electron Paramagnetic Resonance, Western Blot, Immunofluorescence, Staining, Marker, Fluorescence, Microscopy, Labeling, FACS, Flow Cytometry, Transfection, Software

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Nrg1β Released in Remote Ischemic Preconditioning Improves Myocardial Perfusion and Decreases Ischemia/Reperfusion Injury via ErbB2-Mediated Rescue of Endothelial Nitric Oxide Synthase and Abrogation of Trx2 Autophagy

doi: 10.1161/atvbaha.121.315957

Figure Lengend Snippet: Figure 6. Endothelial-ErbB2 (Erb-B2 receptor tyrosine kinase 2) directly associates with Src and activate eNOS (endothelial nitric oxide synthase). A, Human coronary artery endothelial cells (HCAECs) were treated with preconditioned medium (PM) for the indicated period, and cell lysates were prepared. An equal amount of protein from cell lysates was immunoprecipitated using the anti-ErbB2 antibody. The immunoprecipitates were analyzed by Western blotting for tyrosine phosphorylation of ErbB2, level of associated Src, ErbB4, and ErbB2 using their specific antibodies. B, HCAECs were treated with CM for 1 or 4 h, and proximity ligation assay (PLA) was performed using anti-ErbB2 and anti- Src antibodies. Scale bar=20 mm. C, PLA signals were counted and plotted as a bar graph (n=5). *P<0.01 vs control (ANOVA). D, To determine the role of Nrg1 (neuregulin 1) released by HMVEC in PM, PM was pretreated with neutralizing anti-Nrg1 and then incubated with HCAECs for 1 h. At the end of treatment, cell lysates were prepared and analyzed for ErbB2 tyrosine phosphorylation and its interaction with Src immunoprecipitation followed by Western blotting. E, HCAECs were incubated with PM or Nrg1 neutralized-PM for 1 h. and PLA was performed using anti-ErbB2 and anti-Src antibodies. Scale bar=20 mm. F, PLA signals were counted and plotted as a bar graph (n=5). *P<0.01 vs control (ANOVA). G, HCAECs were pretreated with Herceptin and incubated with PM for 1 h, and cell lysates were prepared. An equal amount of proteins from cell lysates were immunoprecipitated with anti-ErbB2 antibodies, and the immunocomplexes were analyzed for associated Src by Western blotting. H, HCAEC were transfected with ErbB2 siRNA, treated with PM for 1 h, and cell lysates were prepared and analyzed for eNOS tyrosine phosphorylation. I, HCAECs were pretreated with control IgG, Herceptin, or Nrg1 neutralizing antibodies and then incubated with PM for 1 h, and cell lysates were prepared. An equal amount of proteins from cell lysates were analyzed for eNOS-Tyr81 phosphorylation by Western blotting using its specific antibodies. J, HCAECs were pretreated with control IgG or Herceptin and incubated with PM for 1 h, and cell lysates were prepared. An equal amount of protein from cell lysates was analyzed for eNOS activity and plotted as a bar graph. *P<0.01 vs control IgG, **P<0.01 vs control IgG+PM (ANOVA).

Article Snippet: Recombinant human Nrg1β1 active domain (catalog No. 396-HB) and neutralizing anti-Nrg1 antibody (catalog No. AF-396-NA) were bought from R&D Systems.

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Proximity Ligation Assay, Control, Incubation, Transfection, Activity Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Nrg1β Released in Remote Ischemic Preconditioning Improves Myocardial Perfusion and Decreases Ischemia/Reperfusion Injury via ErbB2-Mediated Rescue of Endothelial Nitric Oxide Synthase and Abrogation of Trx2 Autophagy

doi: 10.1161/atvbaha.121.315957

Figure Lengend Snippet: Figure 7. Endothelial ErbB2 (Erb-B2 receptor tyrosine kinase 2) dimerizes with ErbB4, recruits Src and mediates Nrg1 (neuregulin 1)-dependent eNOS (endothelial nitric oxide synthase) activation during ischemic preconditioning. A, HCAECs were treated with preconditioned medium (PM) for the indicated period, and cell lysates were prepared. An equal amount of protein from cell lysates was immunoprecipitated using anti-ErbB4 antibody, and the immunoprecipitates were analyzed by Western blotting for tyrosine phosphorylation of ErbB4 and associated ErbB2 levels using their specific antibodies. B, To study the in vivo interaction of ErbB2 and ErbB4, HCAECs were treated with PM for 1 or 4 h, and proximity ligation assay (PLA) was performed using anti-ErbB2 and anti-ErbB4 antibodies. Scale bar=20 mm. C, Green foci-proximity signals of ErbB2 and ErbB2 association were counted and plotted as a bar graph. *P<0.01 vs control (ANOVA). D–F, HCAECs were incubated with PM or Nrg1 neutralized-PM for 1 h, and either cell lysates were prepared and analyzed for ErbB4 tyrosine phosphorylation and its interaction with ErbB2 by Western blotting as described in A or subjected to PLA (E) using anti-ErbB2 and anti-ErbB4 antibodies as described in B. PLA signals were counted and plotted as a bar graph (n=5), Scale bar=20 mm (F). *P<0.01 vs control IgG; **P<0.01 vs neutralizing anti-Nrg1 antibody+PM. G–J, HCAECs were transfected with ErbB4 siRNA, treated with PM for 1 h., and the cell lysates were prepared and analyzed for ErbB2 tyrosine phosphorylation (G), Src activation (H), or eNOS tyrosine phosphorylation (I). An equal amount of protein from cell lysates was analyzed for eNOS activity (J). *P<0.01 vs nontarget (NT) siRNA; **P<0.01 vs NT siRNA+PM (ANOVA). K, To study the role of ErbB2 and ErbB4 in NO formation in coronary arteries, left coronary artery were isolated and incubated with control IgG, neutralizing anti-ErbB2 or neutralizing anti-ErbB4 antibodies, and NO release in response to ACh (10 µmol/L) was determined by electron spin resonance (EPR) using Fe- (MGD)2. L, Spin count of the NO-Fe(MGD)2 was calculated from EPR signals were plotted as a bar graph. *P<0.01 vs control IgG (ANOVA).

Article Snippet: Recombinant human Nrg1β1 active domain (catalog No. 396-HB) and neutralizing anti-Nrg1 antibody (catalog No. AF-396-NA) were bought from R&D Systems.

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Phospho-proteomics, In Vivo, Proximity Ligation Assay, Control, Incubation, Transfection, Activity Assay, Isolation, Electron Paramagnetic Resonance