Methods for studying ER stress and UPR markers in human cells

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Cellular dysfunction often leads to endoplasmic reticulum (ER) stress.
• Accumulation of unfolded proteins in the ER lumen triggers the unfolded protein response (UPR).
• Three key ER-resident transmembrane proteins (PERK, ATF6, IRE1) sense ER stress and initiate UPR signaling.

Purpose of the Study:

• To describe methods for monitoring ER stress and UPR signaling in human cells.
• To analyze activation markers of all three ER stress sensor proteins.
• To provide a comprehensive assessment of UPR activation.

Main Methods:

• Utilizing quantitative PCR (qPCR) for qualitative and quantitative analysis of UPR-induced transcripts.
• Employing Western blot techniques for analyzing UPR-induced proteins and their modifications.
• Combining both methods to assess the activation of PERK, ATF6, and IRE1 signaling pathways.

Main Results:

• Established two distinct sets of methods for UPR detection.
• Demonstrated the ability to analyze UPR markers across all three major signaling pathways.
• Enabled the combined assessment of ER stress sensor activation.

Conclusions:

• The described methods allow for robust monitoring of ER stress and UPR.
• Combined analysis provides a comprehensive view of UPR activation.
• These techniques are valuable for studying cellular responses to ER stress.