Assays for detecting the unfolded protein response.
The endoplasmic reticulum (ER) is the site for folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the ER cause ER stress and activate a set of signaling pathways termed the unfolded protein response (UPR). The UPR leads to transcriptional activation of genes encoding ER-resident chaperones, oxidoreductases, and ER-associated degradation (ERAD) components. Thus, UPR promotes cellular repair and adaptation by enhancing protein-folding capacity, reducing the secretory protein load, and promoting degradation of misfolded proteins. In mammalian cells, the UPR also triggers apoptosis, perhaps when adaptive responses fail. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. Here, we describe the experimental methods that we have used to study UPR in tissue culture cells. These methods can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated or not. It is important to note that these are general guidelines for monitoring the UPR and not all assays will be appropriate for every model system.
Apoptosis Research Centre, School of Natural Sciences (Biochemistry), National University of Ireland Galway, Ireland.
Karen
K
Cawley
Shane
S
Deegan
Afshin
A
Samali
Sanjeev
S
Gupta
D051702:Activating Transcription Factor 6 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000502:physiology; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D013312:Stress, Physiological; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D056811:Unfolded Protein Response / Q000502:physiology*; D019892:eIF-2 Kinase / Q000378:metabolism
D051702:Activating Transcription Factor 6; D004268:DNA-Binding Proteins; D004722:Endoribonucleases; D008565:Membrane Proteins; D018832:Molecular Chaperones; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D019892:eIF-2 Kinase; C497111:ATF6 protein, human; C519634:XBP-1 venus fusion protein; C403982:ERN2 protein, human