Anti-Heparanase 1/HPSE Antibody Picoband®

Western blot analysis of Heparanase 1 using anti-Heparanase 1 antibody (PB9427).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours.
Lane 1: Rat Liver Tissue Lysate at 50ug,
Lane 2: Human Placenta Tissue Lysate at 50ug,
Lane 3: A549 Whole Cell Lysate at 40ug.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Heparanase 1 antigen affinity purified polyclonal antibody (Catalog # PB9427) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for Heparanase 1 at approximately 61 kDa. The expected band size for Heparanase 1 is at 61 kDa.

( a ) Representative images of rat lungs after procurement. ( b ) Heparanase activity was detected in tissue after procurement following 1 h of warm ischemia. ( c ) Ultrastructural images of the endothelial glycocalyx (eGC) from lung grafts after 1 h of warm ischemia obtained using transmission electron microscopy. The labeled glycocalyx appears as the darker cell surface layer; black arrow indicates glycocalyx desquamated from the surface of an endothelial cell. *p < 0.05, **p < 0.01. NL native lungs, Ctrl control lungs with eGC damage induced by 1 h of ischemia, Hep lungs with eGC preserved by heparin administration prior to 1 h of ischemia, NAH lungs with intact eGC preserved by N-acetyl heparin administration prior to 1 h of ischemia.
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The effect of eGC damage in the lung grafts on the posttransplant graft function and quality. Grafts were evaluated 2 h after transplantation for ( a ) PaO2/FiO2 (P/F ratio), ( b ) macroscopic morphology (representative samples are shown), ( c ) Wet-to-dry (W/D) ratio, and ( d ) microscopic morphology (representative H&E stained samples are shown). *p < 0.05, **p < 0.01. NL native lungs, Ctrl control lungs with eGC damage prior to transplant, Hep lungs with eGC preserved by heparin prior to transplant, NAH lungs with eGC preserved by N-acetyl heparin prior to transplant, I/R injury ischemia reperfusion injury.
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Endothelial glycocalyx influences the inflammation and inflammatory cell migration in lung grafts after transplantation. ( a ) Real-time RT-PCR for the mRNA of proinflammatory cytokines interleukin (IL)-6 and IL-1β . ( b ) The extravasation of neutrophils, T cells, and monocytes in the transplanted grafts were evaluated by specific staining of polymorphonuclear neutrophils (naphthol staining, positive cells indicated by black arrow heads), T cells (CD3 + -positive, indicated by white arrows) and macrophages (CD68 + -positive, purple staining), and ( c ) quantitated. *p < 0.05, **p < 0.01. NL native lungs, Ctrl control lungs with eGC damage prior to transplant, Hep lungs with eGC preserved by heparin prior to transplant, NAH lungs with eGC preserved by N-acetyl heparin prior to transplant.
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Assessment of endothelial glycocalyx and microvascular integrity in lung grafts after reperfusion. ( a ) Western blot of syendecan-1 in fractionated lung tissue 2 h after transplantation. Gel image depicts a single representative sample from each treatment group. Vascular endothelial cadherin (VE-Cad) and heat shock protein 90 (HSP90) were blotted as loading markers for the plasma membrane and cytosolic protein fractions, respectively. A full-length image of this blot and the blots showing multiple samples for each treatment group are shown in Supplemental Figs. – . Cyt cytosol protein fraction, M membrane protein fraction. ( b ) Glycosaminoglycan (GAG) content in the grafts 2 h after transplantation. ( c ) Syndecan-1 staining (yellow) in the peripheral vasculature (φ ~ 50 mm) of the grafts 2 h after transplantation. The endothelial luminal surface is indicated as a white line in the merged images (right column). Nuclei were stained using Hoechst 33342. BAF background autofluorescence, RBCs red blood cells, VL vascular lumen. ( d ) Pulmonary vascular resistance (PVR) of lung grafts during ex vivo lung perfusion (EVLP). Time-dependent fold changes from the values at 1 h are shown. n = 4–5 for each group. ( e ) Endothelial barrier integrity after EVLP was assessed by measuring the amount of Evans blue dye (EBD) retained in the tissue. ( f ) Time-dependent changes of syndecan-1 in the perfusate during EVLP. *p < 0.05, **p < 0.01. NL native lungs, Ctrl control lungs with eGC damage prior to transplant/EVLP, Hep lungs with eGC preserved by heparin treatment prior to transplant or EVLP, NAH lungs with eGC preserved by N-acetyl heparin treatment prior to transplant or EVLP.
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The effects of heparanase inhibition on MMP-2 and MMP-9 activity. ( a ) The mRNA expression of metalloprotease (MMP)-9 in lung grafts 2 h after transplantation. ( b ) Representative gelatin zymography of time-dependent changes in activity of secreted MMP-2 and MMP-9 in EVLP perfusate. Full length of gels and replications are shown in Supplemental Fig. . ( c ) MMP-2 activity quantitated and shown as fold change. *p < 0.05, **p < 0.01. NL native lungs, Ctrl control, Hep heparin, NAH N-acetyl heparin.
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Viability analysis of Ana-1 macrophages after treatment with AGEs, LY294002, anti-RAGE or HPA antibody. Cell viability assay is performed using MTT assay. A , Cells (5 × 10 4 ) were treated with AGEs (0, 25, 50, 100, 200 and 400 mg/L) for 3, 6, 12, 24 h. B , Cells (5 × 10 4 ) were pretreated with LY294002 (7.5-120 μM) for 1 h before culture with 100 mg/L AGEs for 3, 6, 12, 24 h. C , Cells (5 × 10 4 ) were pretreated with anti-RAGE or HPA antibody for 1 h before culture with 100 mg/L AGEs for 3, 6, 12, 24 h. The results represent the mean of six culture wells (mean ± SEM). *p < 0.05, as compared to the control group. All of the experiments were performed independently in triplicate.
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HPA, RAGE and PI3K/AKT pathway correlate with AGEs-induced macrophage migration. Cells were cultured with AGEs for 24 h with or without pre-treatment with LY294002, anti-HPA or RAGE antibody for 1 h. The migration was measured by transwell assays. Results were normalized to the number of macrophages that migrated in control group. The results represent the mean of six culture wells (mean ± SEM). *p < 0.05 compared to control and #p <0.05 compared to AGEs. All of the experiments were performed independently in triplicate.
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AGEs up-regulates HPA mRNA, protein expression and secretion in macrophages via RAGE. Cells were cultured with AGEs for 24 h with or without pre-treatment with antibody against RAGE for 1 h. A , The levels of HPA mRNA were assessed with real time quantitative RT-PCR. B , The secretion of HPA in supernatant was measured by enzyme-linked immunosorbent assay (ELISA). C , The expression of HPA protein in macrophages was determined by Western blotting. The results represent the mean of six culture wells (mean ± SEM). *p < 0.05 compared to control and #p <0.05 compared to AGEs. All of the experiments were performed independently in triplicate.
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The expression of AKT protein in AGEs-induced macrophages. Cells were cultured with AGEs or pretreated with antibody against RAGE or HPA for 1 h before exposed to AGEs for 24 h. AKT and p-AKT protein expression is determined by Western blot analysis using anti-AKT and p-AKT antibody. The results represent the mean of six culture wells (mean ± SEM). * P < 0.05 compared to control and # P <0.05 compared to AGEs. All of the experiments were performed independently in triplicate.
Index in PubMed under a CC BY license. PMID: 23442498