Journal: Glycoconjugate Journal

Article Title: Structural characteristics of Heparan sulfate required for the binding with the virus processing Enzyme Furin

doi: 10.1007/s10719-021-10018-8

Figure Lengend Snippet: Furin is a novel heparin/heparan sulfate binding protein. The binding affinity of biotinylated-heparin to immobilized Furin is about 351.5 μg/ml or 23.4 μM based on ELISA a . The binding of Furin to biotinylated-heparin immobilized onto the streptavidin-based probe is determined by biolayer interferometry (BLI) and the K D value is 9.78 nM. The concentration of Furin is 10.2, 20.3, 40.7, 81.3, 325.2, and 650.4 nM b . The binding of Furin or pre-mixture of Furin and heparin (Hep) to murine wild type lung endothelial cells based on cell-based ELISA, the cells without incubation with Furin served as control c . The binding of Furin to murine lung endothelial cells (MLECs) with heparan sulfate (HS) depleted (Ext1 −/− cells) is significantly reduced by about 50% compared to the wild type cells as determined by cell-based ELISA. Heparan sulfate inhibitor surfen (20 μM) or heparinase I, II, and III (HSase, 4 U/ml, 0.5 U/ml, 0.5 U/ml), but not by chondroitinase ABC (CSase ABC, 5 U/ml) treatment at RT for 30 min impairs the binding of Furin to wild type murine lung endothelial cells, the cells without treatment serves as control d . Based on flow cytometry analysis, the binding of Furin to cell surface HS is significantly reduced in the HS depleted cells (Ext1 −/− cells) as the APC median fluorescence intensity (MFI) by anti-His antibody conjugated with APC is significantly reduced e . The bold line and dash line are isotype control for wild type cells and Ext1 −/− cells, respectively; the red and cyan are wild type cells and Ext1 −/− cells, respectively f . Absorbance at 450 nm (OD450) is the indicative of binding capacity. Data are presented as Mean ± SD, ns indicates not significant, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001

Article Snippet: Pierce™ High Sensitivity Streptavidin-HRP (50 ng/ml, Thermo, 21132) was added and incubated for 1 h at RT.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, In-Cell ELISA, Incubation, Flow Cytometry, Fluorescence

Journal: Cancer Science

Article Title: Tuberin regulates reactive oxygen species in renal proximal cells, kidney from rodents, and kidney from patients with tuberous sclerosis complex

doi: 10.1111/cas.12984

Figure Lengend Snippet: Tuberin deficiency increases reactive oxygen species ( ROS ) formation and NADPH ‐dependent oxidase activity in renal primary proximal tubular epithelial ( RPTE ) cells isolated from wild‐type and TSC 2 +/− rats. (a) Intracellular ROS production was measured using the peroxide‐sensitive fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate ( DCF ‐ DA ) in RPTE cells. Deficiency in tuberin significantly increased ROS formation in TSC 2 +/− cells compared to wild‐type cells. The data were quantitated, and the results are expressed as the means ± SE . (b) NADPH oxidase activity measured by lucigenin‐enhanced chemiluminescence in RPTE cell homogenates. Significant increases in NADPH oxidase activity were detected in TSC 2 +/− cells compared to wild‐type cells. Production of NADPH oxidase activity is expressed as relative light units ( RLU )/mg protein/min and normalized as a percentage of the control. ** P < 0.01, significant difference from wild‐type cells. (c) Deficiency in tuberin results in upregulation of NADPH oxidase (Nox) expression in primary culture of RPTE cells. Cell lysates were prepared and protein extracts were loaded onto 7% SDS –polyacrylamide gels and transferred to a PVDF membrane. The membrane was incubated with anti‐tuberin or anti‐Nox1, anti‐Nox2, anti‐Nox4, anti‐P‐p70S6K, and anti‐p70S6K followed by specific HRP ‐conjugated secondary antibodies. Actin was used as a loading control.

Article Snippet: The peroxide‐sensitive fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate (DCF‐DA; Molecular Probes, Carlsbad, CA, USA) was used to assess the generation of intracellular ROS as described previously.

Techniques: Activity Assay, Isolation, Expressing, Incubation

Journal: Cancer Science

Article Title: Tuberin regulates reactive oxygen species in renal proximal cells, kidney from rodents, and kidney from patients with tuberous sclerosis complex

doi: 10.1111/cas.12984

Figure Lengend Snippet: Introduction of TSC 2 cDNA into tuberin‐deficient cells restored the wild‐type pattern of reactive oxygen species ( ROS ), decreased NADPH oxidase (Nox) activity, and abolished Nox isoform expression. Tuberin‐null cells were grown on a 6‐well plate and infected with an adenovirus expressing tuberin (Ad‐ TSC 2 ). An otherwise identical adenovirus expressing β‐Gal (Adβ‐Gal) was used as a control. (a) Intracellular ROS production was measured using the peroxide‐sensitive fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate ( DCF ‐ DA ) in wild‐type and infected null‐tuberin cells with β‐Gal or Ad‐ TSC 2 ( AD 6.01). The data were quantitated and the results are expressed as the means ± SE . (b) NADPH ‐dependent ROS generation measured by lucigenin‐enhanced chemiluminescence in wild‐type and infected null‐tuberin cells with β‐Gal or Ad‐ TSC 2 ( AD 6.01) homogenates. Production is expressed as relative light units ( RLU )/mg protein/min and normalized as a percentage of the control. ** P < 0.01, significant difference from wild‐type cells. (c) Introduction of TSC 2 cDNA into tuberin‐deficient cells abolished Nox protein expression. Cell lysates of wild‐type and infected null‐tuberin cells with β‐Gal or Ad‐ TSC 2 ( AD 6.01) were prepared. Western blot was carried out and the membrane was incubated with antibody specific for tuberin, Nox2, and Nox4, followed by HRP ‐conjugated secondary antibody. The proteins were visualized with ECL . Actin expression served as a loading control. (d–f) Rapamycin blocks activation of mammalian target of rapamycin ( mTOR ) activation to decrease expression of Nox subunits in mouse embryonic fibroblast ( MEF ) cells. MEF cells were treated with different concentrations of rapamycin (0–100 nM) for 24 h. MEF cell lysates of TSC 2 +/+ , TSC 2 +/− , and TSC 2 −/− were prepared and protein extracts and analyzed by Western blot. Data showed a slight decrease in Nox1 in TSC 2 +/− cells treated with 100 nM rapamycin. Rapamycin treatment at concentrations 40–100 nM showed a decrease in protein expression of Nox2. In addition, the higher concentration of rapamycin (40 nM) showed a significant decrease in Nox4 in TSC 2 +/− and TSC 2 −/− cells. (g, h) Tuberin‐deficient cells expressed higher protein kinase C ( PKC ) protein; treating the cells with PKC inhibitor significantly decreased Nox protein expression. (g) Significant increase in PKC β II expression was detected in TSC 2 −/− and TSC 2 +/− cells compared to TSC 2 +/+ cells by Western blot analysis. (h) TSC 2 −/− cells showed higher expression of all Nox isoforms were treated with 2.5 and 5.0 nm of bisindolylmaleimide I ( BMI ; PKC inhibitor) for 24 h before harvesting for Western blot analysis. Data showed significant decrease in the protein expression of Nox1, Nox2, and Nox4 compared to non‐treated cells, suggesting that PKC is an upstream target of Nox. GAPDH was used as loading control.

Article Snippet: The peroxide‐sensitive fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate (DCF‐DA; Molecular Probes, Carlsbad, CA, USA) was used to assess the generation of intracellular ROS as described previously.

Techniques: Activity Assay, Expressing, Infection, Western Blot, Incubation, Activation Assay, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: NOX1 promotes myocardial fibrosis and cardiac dysfunction via activating the TLR2/NF-κB pathway in diabetic cardiomyopathy

doi: 10.3389/fphar.2022.928762

Figure Lengend Snippet: NOX1 promoting fibrosis and oxidative stress in CFs. (A,G) Representative image of α-SMA expression (red) in myocardial cells by immunofluorescence. The nuclei were identified by DAPI (blue). (B,H) Gene expression levels of Col I, Col III, α-SMA, and TGF-β1. (C,I) Protein levels of Col I, Col III, α-SMA, and TGF-β1, and quantitative analysis in each group. (D,J) Intracellular ROS level was detected by DCFH-DA staining. (E,K) Quantitative result of the ROS level. (F,L) Level of MDA in the indicated group ( n = 3 per group). * for p ≤ 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001.

Article Snippet: Peroxide-sensitive fluorescent probe DCFH-DA (Beyotime, China) was used to detect ROS intracellular levels.

Techniques: Expressing, Immunofluorescence, Staining