Color Atlas of Human Anatomy by Kahle W., Leonhardt H., Platzer W.

By Kahle W., Leonhardt H., Platzer W.

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Incubation temperature is dependent on the restriction enzymes used. 18. Run undigested plasmid DNA on the same gel as a control for restriction enzyme activity. 19. Ligation reactions are generally carried out with an excess of insert DNA. A 3:1 molar ratio of insert to vector is commonly used, but this ratio can be varied from 10:1 to 1:1. 20. Include a negative control ligation in which the insert is replaced by water. This will determine the amount of background colonies formed as a result of incomplete vector digestion or vector religation.

In this article, we present a detailed protocol for the quantification of in vivo ubiquitination and turnover of IκB-α in response to a variety of cellular stimuli. Key words NF-κB, RelA, IκB, Immunoprecipitation, Ubiquitin, Ubiquitination, Ubiquitin-like proteins (UBL) 1 Introduction The NF-κB transcription factors regulate expression of a large number of genes, which in turn drive a variety of physiological processes including innate and adaptive immunity, apoptosis, and cellular differentiation [1].

A) The dynamic of phosphorylation and degradation of IκB-α in total lysates. (b) The signal dependent ubiquitination of IκB-α associate with RelA. Maximal ubiquitination rate is observed between 10 and 15 min of stimulation and after dramatically decreased due to proteasomal degradation of IκB-α (30 min sample). RelA serves as loading controls. 1 Petro Starokadomskyy and Ezra Burstein Materials Reagents 1. Anti-RelA antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). 2. Anti-phospho-IκB-α antibody Signaling, Danvers, MA, USA).

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