Detection and analysis of protein ISGylation.
ISG15 is a ubiquitin-like modifi er that is conjugated to target proteins by a sequential reaction catalyzed by E1/E2/E3 enzymes (protein ISGylation). ISG15 and protein ISGylation are upregulated by interferon stimuli. ISG15 functions as an antiviral protein against Sindbis virus and HIV-1, but the molecular mechanism remains unknown. Here we describe in detail methods for detecting and analyzing protein ISGylation. The methods consist of plasmid transfection and affi nity purifi cation of ISGylated proteins. In addition, we describe a method for detecting ISGylation of a target protein, Ubc13.
Takeuchi
Tomoharu
T
Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Yokosawa
Hideyoshi
H
D015153:Blotting, Western; D002846:Chromatography, Affinity; D016207:Cytokines / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D047468:Immunoprecipitation; D011506:Proteins / Q000378:metabolism*; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism*
D016207:Cytokines; D011506:Proteins; D014452:Ubiquitins; C074034:ISG15 protein, human