Methods for Monitoring Endoplasmic Reticulum Stress and the Unfolded Protein Response
Figure 1
Detection of transcript levels of UPR target genes by RT-PCR. (a) Upper panel, cartoon of XBP1 splicing during ER stress. Lower panel, schematic representation of various mutant constructs of IRE1. (b) Modulation of XBP1 splicing by mutant IRE1. Total RNA was isolated from HEK 293 cells that were transfected with IRE1 mutants, either untreated or treated with thapsigargin (0.5 M) 6 hours, and RT-PCR analysis of total RNA was performed to simultaneously detect both spliced and unspliced XBP1 mRNA and GADPH. (c) Induction of UPR target genes upon exposure to thapsigargin. Total RNA was isolated from indicated cells after treatment thapsigargin (Tg), and the expression levels of the indicated genes were determined by real-time RT-PCR, normalizing against GAPDH expression.
