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Title: In Vitro Activity Assays to Quantitatively Assess the Endogenous Reversible Oxidation State of Protein Tyrosine Phosphatases in Cells
Abstract

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

 
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NSF-PAR ID:
10187736
Author(s) / Creator(s):
 ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Current Protocols in Chemical Biology
Volume:
12
Issue:
3
ISSN:
2160-4762
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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