Assays for detecting the unfolded protein response

Methods Enzymol. 2011;490:31-51. doi: 10.1016/B978-0-12-385114-7.00002-7.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Activating Transcription Factor 6 / genetics
  • Activating Transcription Factor 6 / metabolism
  • Animals
  • Biological Assay / instrumentation
  • Biological Assay / methods*
  • Cells, Cultured
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Endoplasmic Reticulum / physiology
  • Endoribonucleases / genetics
  • Endoribonucleases / metabolism
  • Gene Expression Profiling
  • Genes, Reporter
  • Humans
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods
  • Models, Biological
  • Molecular Chaperones / metabolism
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • RNA Splicing
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Regulatory Factor X Transcription Factors
  • Stress, Physiological
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Unfolded Protein Response / physiology*
  • eIF-2 Kinase / metabolism

Substances

  • ATF6 protein, human
  • Activating Transcription Factor 6
  • DNA-Binding Proteins
  • Membrane Proteins
  • Molecular Chaperones
  • Recombinant Fusion Proteins
  • Regulatory Factor X Transcription Factors
  • Transcription Factors
  • XBP-1 venus fusion protein
  • ERN2 protein, human
  • PERK kinase
  • Protein-Serine-Threonine Kinases
  • eIF-2 Kinase
  • Endoribonucleases