title: Determination of protein turnover rates in the JAK/STAT pathway using a radioactive pulse-chase approach.
abstract: The turnover rate of different protein species in a signal transduction network strongly affects the impact of the given species on the outcome of a stimulus. Whereas stable, long-lived proteins mainly account for the transmission of a signal, unstable short-lived species often comprise regulatory functions. Here, we describe a method to determine the half-lives of proteins of the JAK/STAT pathway by a pulse-chase approach in cell culture. First, radioactive labeling with (35)S-methionine is carried out to label newly synthesized proteins (pulse). Subsequently, the dynamics of the decay of these proteins is monitored in the absence of labeled amino acids over a defined time period (chase). For this purpose the protein of interest is isolated by immunoprecipitation from total cell lysates, separated on an SDS-polyacrylamide gel, and subsequently visualized by autoradiography.
authors.affiliation: Department of Systems Biology, Institute of Biology, Otto-von-Guericke-University, Magdeburg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Dittrich
authors.forename: Elmar
authors.initials: E
authors.lastname: Siewert
authors.forename: Fred
authors.initials: F
authors.lastname: Schaper
mesh_terms: D000818:Animals; D001345:Autoradiography; D019556:COS Cells; D002457:Cell Extracts; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D006207:Half-Life; D056945:Hep G2 Cells; D006801:Humans; D047468:Immunoprecipitation; D053612:Janus Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D050791:STAT Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction; D013194:Staining and Labeling; D013462:Sulfur Radioisotopes; D014162:Transfection
chemical_list: D002457:Cell Extracts; D050791:STAT Transcription Factors; D013462:Sulfur Radioisotopes; D053612:Janus Kinases