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Methods Mol Biol ; 1982: 153-171, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31172472

RESUMEN

Structure-function analysis of specific regions of NOX2 can be carried out after stable expression of site-directed mutagenesis-modified NOX2 in the X0-CGD PLB-985 cell model. Indeed, the generation of this human cellular model by Prof. MC Dinauer's team gave researchers the opportunity to gain a deeper understanding of functional regions of NOX2. With this model cell line, the functional impact of X+-CGD or of new mutations in NOX2 can be highlighted, as the biological material is not limited. PLB-985 cells transfected with various NOX2 mutations can be easily cultured and differentiated into neutrophils or monocytes/macrophages. Several measurements in intact mutated NOX2 PLB-985 cells can be carried out such as NOX2 expression, cytochrome b 558 spectrum, enzymatic activity, and assembly of the NADPH oxidase complex. Purified membranes or purified cytochrome b 558 from mutated NOX2 PLB-985 cells can be used for the study of the impact of specific mutations on NADPH oxidase or diaphorase activity, FAD incorporation, and NADPH or NADH binding in a cell-free assay system. Here, we describe a method to generate mutated NOX2 PLB-985 cells in order to analyze NOX2 structure-function relationships.


Asunto(s)
NADPH Oxidasa 2/química , NADPH Oxidasa 2/metabolismo , Línea Celular , Clonación Molecular , ADN Complementario , Activación Enzimática , Citometría de Flujo , Expresión Génica , Granulocitos/metabolismo , Humanos , Mediciones Luminiscentes , Mutagénesis Sitio-Dirigida , NADPH Oxidasa 2/genética , Plásmidos/genética , Proteínas Recombinantes , Relación Estructura-Actividad
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