Analysis of ubiquitin degradation and phosphorylation of proteins.
Both ubiquitination and phosphorylation are crucial mediators involved in controlling the functions of numerous proteins belonging to the Toll-like receptor (TLR) signaling pathways. Altering the aforementioned post-translational events can be detrimental to the host survival. Therefore, the importance of these modifications cannot be overestimated. This chapter describes techniques used to examine if a protein is ubiquitinated and/or phosphorylated. In addition, a method is provided to identify the modified amino acids. We have previously shown using these techniques that the protein MyD88 adapter-like (Mal) is phosphorylated and ubiquitinated following activation of the TLR2 and TLR4 signaling pathways. Both post-translational modifications are essential for the activation and degradation of Mal, and thus are crucial steps, in regulating these TLR signaling cascades and consequently the innate immune response.
Gray
Pearl
P
Division of Pediatric Infectious Diseases, University of California Los Angeles, Los Angeles, CA 90048, USA. grayp@cshs.org
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007422:Intestines / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D011506:Proteins; D000469:Alkaline Phosphatase