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Identification of Factors in Regulating a Protein Ubiquitination by Immunoprecipitation: a Case Study of TRF2 on Human REST4 Ubiquitination.

Bio-protocol (2015-07-05)
Patrick Ovando-Roche, Wei Cui
ABSTRACT

Ubiquitination is the first step of the ubiquitin-proteasome pathway that regulates cells for their homeostatic functions and is an enzymatic, protein post-translational modification process in which ubiquitin is transferred to a target protein substrate by a set of three ubiquitin enzymes (Weissman et al., 2011; Bhattacharyya et al., 2014; Ristic et al., 2014). Given the importance of this process, it is plausible that ubiquitination is under strict control by many factors and that the regulatory machineries are protein-specific. An assay for the detection of a specific protein ubiquitination will enable us to examine whether a factor has a function to regulate the ubiquitination of this protein. Here we describe a protocol that detects the ubiquitination status of the human REST4 protein in cultured cells, a neural alternative splicing isoform of REST (RE-1 silencing transcription factor), that antagonizes the repressive function of REST on neural differentiation and neuron formation. Using this protocol, we show that the telomere binding protein TRF2 stabilizes the expression of the human REST4 by inhibiting its ubiquitination. This indicates that TRF2 plays a positive role in neural differentiation (Ovando-Roche et al., 2014). This protocol is also useful for the detection of ubiquitination of other proteins of interest.

MATERIALS
Product Number
Brand
Product Description

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Monoclonal Anti-β-Actin antibody produced in mouse, clone AC-15, ascites fluid
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Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, ≥97.0%
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Calcium Phosphate Transfection Kit, Most cost effective transfection reagent kit for transient and stable transfection of DNA into mammalian cells
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Anti-TRF2 Antibody, clone 4A794, clone 4A794, Upstate®, from mouse
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