In vitro Activity Assays to Quantitatively Assess the Endogenous Reversible Oxidation State of Protein Tyrosine Phosphatases in Cells
| Autor: | Benoit Boivin, Syed Husain Mustafa Rizvi, Avinash D. Londhe |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
chemistry.chemical_classification Reactive oxygen species Chemistry General Medicine Protein tyrosine phosphatase Transfection 010402 general chemistry 01 natural sciences In vitro Article 0104 chemical sciences Cell biology 03 medical and health sciences 030104 developmental biology HEK293 Cells In vivo Biotinylation Humans Signal transduction Protein Tyrosine Phosphatases Oxidation-Reduction Cysteine |
| Zdroj: | Curr Protoc Chem Biol |
| Popis: | The reversible oxidation of protein tyrosine phosphatases (PTPs) impairs their ability to dephosphorylate substrates in vivo. This transient inactivation of PTPs occurs as their conserved catalytic cysteine residue reacts with cellular oxidants thereby abolishing the ability of this reactive cysteine to attack the phosphate of the target substrate. Hence, in vivo, the inhibition of specific PTPs in response to regulated and localized rises in cellular oxidants enables phospho-dependent signaling. We present assays that measure the endogenous activity of specific PTPs that become transiently inactivated in cells exposed to growth factors. Here, we describe the methods and highlight the pitfalls to avoid post-lysis oxidation of PTPs in order to assess the inactivation and the reactivation of PTPs targeted by cellular oxidants in signal transduction. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Cell transfection (optional) Support Protocol: Preparation of degassed lysis buffers Basic Protocol 2: Cellular extraction in anaerobic conditions Basic Protocol 3: Enrichment and activity assay of specific PTPs Alternate Protocol: Measurement of active PTPs via direct cysteinyl labeling. |
| Databáze: | OpenAIRE |
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