Protein Detection by Simple Western™ Analysis

Harris, Valerie M.

doi:10.1007/978-1-4939-2694-7_47

Cited by 109 publications

(80 citation statements)

References 3 publications

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“…Likewise, because prostasin is heterogeneously glycosylated and undergoes C-terminal processing, and because activation site cleavage and reduction of prostasin leads to the removal of just 12 amino acids [39], analysis of the state of activation of prostasin proved difficult using standard techniques. We therefore employed the Peggy Sue capillary electrophoresis system, which separates proteins by size with high resolution and detects them by immunoassay in a sensitive and quantitative format [40], to examine the expression of matriptase and prostasin in the gene-edited mice. To aid the identification of the various forms of prostasin, we included in parallel skin from mice expressing zymogen-locked endogenous prostasin [41].…”

Section: Resultsmentioning

confidence: 99%

“…The Peggy Sue capillary electrophoresis system [40] (Protein Simple, Wallingford, CT, USA) was used to analyze matriptase and prostasin in lysates from newborn mice. Briefly, snap frozen skin, kidney, lung or intestines from newborn mice were lysed in T-PER lysis buffer with proteinase and phosphatase inhibitors (Pierce) as well as the serine protease inhibitor AEBSF to a final concentration of 0.5 mM (Sigma).…”

Section: Methodsmentioning

confidence: 99%

“…Quantitative analysis of immunoreactive proteins was performed using the Compass Software, which automatically measures the luminescence signal as a function of the amount of protein present in the sample at a certain molecular mass. The software calculates the area under the curve as a measure of the amount of protein [40]. …”

Section: Methodsmentioning

confidence: 99%

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Matriptase zymogen supports epithelial development, homeostasis and regeneration

et al. 2017

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Background: Matriptase is a membrane serine protease essential for epithelial development, homeostasis, and regeneration, as well as a central orchestrator of pathogenic pericellular signaling in the context of inflammatory and proliferative diseases. Matriptase is an unusual protease in that its zymogen displays measurable enzymatic activity. Results: Here, we used gain and loss of function genetics to investigate the possible biological functions of zymogen matriptase. Unexpectedly, transgenic mice mis-expressing a zymogen-locked version of matriptase in the epidermis displayed pathologies previously reported for transgenic mice mis-expressing wildtype epidermal matriptase. Equally surprising, mice engineered to express only zymogen-locked endogenous matriptase, unlike matriptase null mice, were viable, developed epithelial barrier function, and regenerated the injured epithelium. Compatible with these observations, wildtype and zymogen-locked matriptase were equipotent activators of PAR-2 inflammatory signaling. Conclusion: The study demonstrates that the matriptase zymogen is biologically active and is capable of executing developmental and homeostatic functions of the protease.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Matriptase zymogen supports epithelial development, homeostasis and regeneration

et al. 2017

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“…Equivalents of 10 μg proteins were separated on 4–20% SDS-PAGE and subjected to traditional Western blotting. Additionally, equivalents of 1 μg proteins were analyzed with ProteinSimple Wes automated Western blotting system (Harris, 2015). The following antibodies were utilized: mouse monoclonal anti -p53 (Cell Signaling), rabbit polyclonal anti -p53 (Ser 37) (Cell Signaling Technology: cat.…”

Section: Methodsmentioning

confidence: 99%

p53 Modulates the Fate of Cardiac Progenitor Cells Ex Vivo and in the Diabetic Heart In Vivo

et al. 2017

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p53 is an important modulator of stem cell fate, but its role in cardiac progenitor cells (CPCs) is unknown. Here, we tested the effects of a single extra-copy of p53 on the function of CPCs in the presence of oxidative stress mediated by doxorubicin in vitro and type-1 diabetes in vivo. CPCs were obtained from super-p53 transgenic mice (p53-tg), in which the additional allele is regulated in a manner similar to the endogenous protein. Old CPCs with increased p53 dosage showed a superior ability to sustain oxidative stress, repair DNA damage and restore cell division. With doxorubicin, a larger fraction of CPCs carrying an extra-copy of the p53 allele recruited γH2A.X reestablishing DNA integrity. Enhanced p53 expression resulted in a superior tolerance to oxidative stress in vivo by providing CPCs with defense mechanisms necessary to survive in the milieu of the diabetic heart; they engrafted in regions of tissue injury and in three days acquired the cardiomyocyte phenotype. The biological advantage provided by the increased dosage of p53 in CPCs suggests that this genetic strategy may be translated to humans to increase cellular engraftment and growth, critical determinants of successful cell therapy for the failing heart.

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“…Isolated PBMCs and neutrophils were lysed with 100 µl RIPA buffer supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany). Then, cell debris was removed by centrifugation for 10min at 16000 g. The assay steps were carried out according to the instructions recommended by the manufacturer [19] and all the targeted proteins were measured simultaneously to ensure comparable data.…”

Section: Methodsmentioning

confidence: 99%

A Role for Receptor-Interacting Protein Kinase-1 in Neutrophil Extracellular Trap Formation in Patients with Systemic Lupus Erythematosus: a Preliminary Study

Guo

Xie

et al. 2018

Cell Physiol Biochem

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Background/Aims: Neutrophil extracellular traps (NETs) are known to play an important role in systemic lupus erythematosus (SLE) by triggering innate and adaptive immune responses. The molecular mechanisms responsible for their formation in SLE are still unclear. In this study, we aim to characterize the role of the receptor-interacting protein kinase-1 (RIPK1), a homologous serine/threonine kinase previously implicated in the regulation of necroptosis and tissue injury, in decreasing neutrophil death and formation of NETs, and to investigate the clinical implications of RIPK1 in SLE. Methods: Patients with SLE (n = 50) and healthy donors (n = 35) were enrolled in in vitro studies. Management of SLE patients was evaluated using the SLE disease activity index 2000 (SLEDAI-2K) score and laboratory variables. The mRNA level of RIPKs was measured by quantitative polymerase chain reaction (qPCR). Intracellular RIPK1 and RIPK3 production by peripheral blood leukocytes was detected by four-color flow cytometry and confirmed by automatic western blotting. TNF-α, IFN-γ, IL-1β, IL-2, IL-8, IL-18, and RIPK1 were measured by enzyme-linked immunosorbent assay. Cell death was assayed by Sytox green dye from peripheral neutrophils stimulated by RIPK-1-stabilizer necrostatin-1 (nec-1) and phorbol 12-myristate 13-acetate (PMA). Immunofluorescence staining and confocal microscopy were used to detect NET formation ex vivo. Quantification of NETs was determined by fluorescence spectrometry. Results: IFN-γ, IL-1β, IL-8, and IL-18 levels in serum were increased in SLE patients compared to controls. However, the expression of TNF-α, IL-2, and RIPK1 were decreased. In addition, we observed significant differences in the expression of RIPK1 in peripheral blood leukocytes. Of all the leukocytes, RIPK1 expression was significantly lower in neutrophils. Furthermore, we studied NETs formation in neutrophils of SLE with decreased RIPK1 expression, and these show increased susceptibility to NETosis, when stimulated with PMA and/or nec-1. Importantly, RIPK1 expression in neutrophils negatively correlated with ESR, CRP, 24-hour urine total protein, and the disease activity index in SLE. Conclusion: These data represent the first report of decreased RIPK1 expression in neutrophils of SLE patients and imply that RIPK1 may be involved in neutrophil death and NET formation. We suggest that RIPK1 is a potential biomarker to predict disease activity.

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Protein Detection by Simple Western™ Analysis

Cited by 109 publications

References 3 publications

Matriptase zymogen supports epithelial development, homeostasis and regeneration

Matriptase zymogen supports epithelial development, homeostasis and regeneration

p53 Modulates the Fate of Cardiac Progenitor Cells Ex Vivo and in the Diabetic Heart In Vivo

A Role for Receptor-Interacting Protein Kinase-1 in Neutrophil Extracellular Trap Formation in Patients with Systemic Lupus Erythematosus: a Preliminary Study

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