Endoplasmic reticulum stress will activate a signaling network called the unfolded protein response (UPR) to reduce this stress and restore endoplasmic reticulum homeostasis, promoting cell survival and adaptation. Because secretion underlies multicellularity, the balance of ER protein folding dynamics has a significant impact on mammalian physiology. Dysregulation of endoplasmic reticulum homeostasis can lead to chronic diseases in humans. Therefore, detection of UPR is essential for the assessment of endoplasmic reticulum stress.

Fig. 1 Studying ER stress and the Unfolded Protein Response in Mammalian cells (Oslowski et al., 2011)

Measuring IRE1α and PERK phosphorylation and the ability of ATF6α cleavage would be ideal to determine the level of UPR activation. However, the endogenous expression levels of these molecules are low and difficult to detect with available commercial antibodies. Creative Bioarray measures the expression and activation levels of downstream components regulated by these master regulators to determine UPR activation.

IRE1α Activation Assay Service

• Measuring the activation level of IRE1α by measuring its phosphorylation level

We can measure IRE1α activation directly by assessing the phosphorylation status of serine 724 or 729 which is phosphorylated upon activation of IRE1α. We typically use commercially available specific antibodies, or we can use customer-supplied antibodies upon request. Because of the low levels of IRE1, we will concentrate the protein by immunoprecipitation before immunoblotting.

• Direct visualization and quantitative analysis services for IRE1 signal

We use the Postage-based WB method, which allows direct visualization and quantification of the IRE1 signal under a variety of conditions.

• Activity testing of the oligomeric state of IRE1

The oligomerization state of IRE1 can be tested to measure its activity, for example, using a super folder green fluorescent protein (GFP)-IRE1 construct in which sfGFP is N-fused to the luminal domain of IRE1.

PERK Activation Assay Service

Similar to IRE1α, PERK undergoes trans-autophosphorylation in response to ER stress. To measure PERK activation, Creative Bioarray detects PERK and eIF2α phosphorylation status using antibodies against total and phosphorylation-specific proteins. the level of PERK phosphorylation can be detected by phosphorylation-specific PERK antibodies. Upon activation, PERK phosphorylates eIF2α to reduce overall mRNA translation. PERK activation is indirectly reflected by measuring eIF2α phosphorylation levels by immunoblotting using anti-photo-eIF2α-specific antibodies.

ATF6α Activation Assay Service

Creative Bioarray detects cleaved 50-kDa forms of ATF6α by immunoblotting using anti-ATF6α-specific antibodies that can be used as indicators of ATF6α activation. Our immunoblotting protocol includes a post-containment exposure step to reveal the antigen used for antibody binding.

List of Antibodies for Detecting IRE1α, PERK, and ATF6α Markers

UPR Downstream Marking Measuring Service

• Immunostaining and immunofluorescence for downstream markers of the UPR
• Measuring transcriptional activation of the UPR
• Measuring translational attenuation of the UPR
• Measuring ERAD and protein stability

Creative Bioarray is dedicated to providing high-quality products, comprehensive services, and tailored solutions to support and facilitate life sciences and pharmaceutical research and development. If you have any questions or needs, please contact us, and our customer service staff will help you the first time.

Reference

1. Oslowski, C. M., & Urano, F. (2011). Measuring ER stress and the unfolded protein response using mammalian tissue culture system. Methods in enzymology, 490, 71–92.

Services

• Cell Level Oxidative Stress Assay Services
  • DNA & RNA Oxidative Stress Detection
  • Lipid Peroxidation Stress Assay
    • Lipid Peroxidation By-products Assay
    • Low Density Lipoprotein Oxidative Stress Assay Service
    • High Density Lipoprotein Oxidative Stress Assay Service
  • Protein Oxidation and Nitration Assays
  • Reactive Oxygen Species Assays
    • Hydrogen Peroxide Assays
    • Nitric Oxide Assays
    • Intracellular ROS Assay
    • In Vitro ROS & RNS Assay
  • Cell Antioxidant Assay Services
    • Antioxidant Capacity Assays Services
    • Antioxidant Enzyme Activity Assays
  • Oxidase & Peroxidase Activity Assay Service
  • Stressed Cell Migration Assay
  • Oxidative Stress Induced Apoptosis Assay Services
  • Stressed Cell Viability and Proliferation Assay
  • Immunohistochemistry Detection Service of ROS
  • Blood OS State Assay Services
  • Urine OS State Assay Services
• Oxidative Stress Cell Model Establishment Services
  • Establishment of Stress Model by Chemical Method
    • Extracellular H₂O₂ Exposure Model System
  • Hyperoxia Cell Oxidative Model System
  • Quality Evaluation Index of Oxidative Stress Cell Model
  • Digestive System Stress Model
    • Intestinal Epithelial Oxidative Stress Cell Model Construction Service
      • Intestinal Epithelial Cell Modeling Methods
      • Intestinal Epithelial Cell for Oxidative Stress Modeling
    • Hepatic Stellate Cell Oxidative Stress Model
      • Rat Hepatic Stellate Cells Oxidative Stress Model
      • Human Hepatic Stellate Cell Oxidative Stress Model
    • Hepatoma Cell Oxidative Stress Model
    • Hepatocyte Oxidative Stress Model
      • Human Hepatocyte Stress Model
      • Hepatocyte Mitochondrial Stress Autophagy Model
      • Chicken Hepatocyte Oxidative Stress Model
  • Reproductive System Oxidative Stress Model
    • Rat Ovarian Oxidative Stress Model
    • Mouse Ovarian Oxidative Stress Model
    • Human Ovarian Granulose Cells Oxidative Stress Model
    • Male Reproductive Cell Oxidative Stress Model
  • Endoplasmic Reticulum Stress Model
    • Liver Cancer Cells ER Stress Model
    • Rhinovirus Vector for ER Stress Modeling
    • Osteoblast ER Stress Model
    • Hepatocyte ER Stress Model
    • Cardiomyocyte ER Stress Model
  • Establishment of Stress Model by Biological Method
    • Porcine Circovirus Type 2 Induction
  • Skeletal System Oxidative Stress Modeling Service
    • Xanthine-Xanthine Oxidase Modeling Service for Osteoblast
    • H₂O₂ Oxidase Modeling Service for Osteoblast
    • Fluoride Oxidase Modeling Service for Osteoblast
    • Chondrocyte Biological Oxidative Stress Modeling Service
    • Chondrocyte Chemical Oxidative Stress
    • Osteoblast Oxidative Stress Level Evaluating Service
    • Skeletal System Antioxidant Screening Service
    • Skeletal System Antioxidant Candidate Evaluating Service
  • Circulatory System Oxidative Stress Modeling Service
    • Myocardial Oxidative Stress
      • Establishment of Myocardial Oxidative Stress Model
      • Cardiomyocyte Antioxidant Evaluation Service
      • Medicine Cardiac Toxicity Assessment Service
      • Gene-Level Stress Assessment of Cardiomyocyte
    • Umbilical Vein Endothelial Cell Oxidative Stress Modeling Service
    • Umbilical Vein Endothelial Cell Antioxidant Evaluation Service
  • Nervous System Oxidative Stress Modeling Service
    • Neural Cell Oxidative Stress Modeling Service
    • Neuroblastoma Oxidative Stress Modeling Service
    • Glioblastoma Oxidative Stress Modeling Service
    • Oxidation Neural Cell Gene Expression Analysis Service
    • Nervous System Antioxidant Candidate Assessment Service
    • Neural Cell Oxidation State Regulation Service
  • Skin System Oxidative Stress Modeling Service
    • Skin Fibroblast Oxidative Stress Service
      • Efficacy Evaluation Service for Anti-aging Substances
      • Regulation of Stress State in Skin Fibroblasts
    • Melanocyte Oxidative Stress Service
  • Animal Stress Modeling and Evaluation Services
    • Animal Oxidative Stress Modeling and Evaluation Services
      • Chicken Embryo Oxidative Stress Modeling Service
      • Pig Oxidative Stress Modeling Service
    • Animal Immune Stress Modeling and Evaluation Services
• Cell Oxidative Stress Assay Service Selection Guide
• Organelle Level Stress Assay
  • Mitochondrial Stress Test
    • Mitochondrial Oxidative Stress State Analysis
    • Mitochondrial Toxicity Assay
      • Functional Mitochondrial Toxicity Assay
      • Mitochondrial Respiratory Complex Assay
      • Mitochondrial Biogenesis Assay
      • HCS Mitochondrial Toxicity Assay
    • ROS Localization Services in Mitochondria
  • Endoplasmic Reticulum Stress Test
    • Continuous Analysis of ER Stress
    • Unfolded Protein Response Assay
    • Rapid Detection and Quantification Service of ER Stress Model
    • ER Stress Induction Platform
    • ER Redox State Monitoring
    • ER Calcium Distribution Measurement
    • Chemical and Genetic Tools to Assess ER Stress
  • Lipid Membrane Stress Test
• Non-oxidative Cellular Stress Assay
  • Hypoxia Stress Molecular Test Services
  • Heat Stress Molecular Test Services