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. 2010 Jul 16;5(7):e11621.
doi: 10.1371/journal.pone.0011621.

A Phos-tag-based approach reveals the extent of physiological endoplasmic reticulum stress

Liu Yang  1 , Zhen Xue, Yin He, Shengyi Sun, Hui Chen, Ling Qi
Affiliations

A Phos-tag-based approach reveals the extent of physiological endoplasmic reticulum stress

Liu Yang et al. PLoS One. .

Abstract

Cellular response to endoplasmic reticulum (ER) stress or unfolded protein response (UPR) is a key defense mechanism associated with many human diseases. Despite its basic and clinical importance, the extent of ER stress inflicted by physiological and pathophysiological conditions remains difficult to quantitate, posing a huge obstacle that has hindered our further understanding of physiological UPR and its future therapeutic potential. Here we have optimized a Phos-tag-based system to detect the activation status of two proximal UPR sensors at the ER membrane. This method allowed for a quantitative assessment of the level of stress in the ER. Our data revealed quantitatively the extent of tissue-specific basal ER stress as well as ER stress caused by the accumulation of misfolded proteins and the fasting-refeeding cycle. Our study may pave the foundation for future studies on physiological UPR, aid in the diagnosis of ER-associated diseases and improve and facilitate therapeutic strategies targeting UPR in vivo.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Visualization and quantitation of ER stress under pharmacological stress.
(A) Immunoblots of IRE1α (upper) and PERK (lower) proteins in Tg-treated MEFs treated with or without λPPase or CIP. (B and D) Immunoblots of IRE1α (B) and PERK (D) using the Phos-tag vs. regular gels. MEFs were treated with 75 nM Tg at indicated period of time. (C) Quantitation of percent of phosphorylated IRE1α in total IRE1α protein in Phos-tag gels shown in B. (E) Immunoblots of IRE1α and PERK in wildtype MEFs treated with Tg at indicated concentrations for 4 h. (F) Quantitation of percent of phosphorylated IRE1α in total IRE1α protein in Phos-tag gels in E. HSP90 and CREB, loading controls. Phos-tag gels are indicated with a bar at the left-hand side. “0” refers to the non- or hypophosphorylated forms of the protein whereas “p” refers to the phosphorylated forms of the protein.
Figure 2
Figure 2. Accumulation of misfolded proteins induces mild ER stress.
(A and C) Immunoblots of IRE1α and PERK in HEK293T cells transfected with the indicated plasmids for 24 h. NHK, the unfolded form of α1-antitrypsin; p97-QQ, dominant negative form of p97-WT. ER-dsRed and GFP, negative control plasmids. HSP90, a position and loading control. (B and D) Quantitation of percent of phosphorylated IRE1α in total IRE1α protein in Phos-tag gels shown in A, C. Values are mean ± SEM *, P<0.05 using unpaired two-tailed Student's t-test. Representative data from at least three independent experiments shown.
Figure 3
Figure 3. Many tissues exhibit basal ER stress under feeding conditions.
(A) Immunoblots of IRE1α and PERK in various tissues of wildtype mice. WAT, white adipose tissues; Panc, pancreas; Muscle, gastrocnemius. HSP90, a position and loading control. (B–C) Immunoblots of IRE1α and PERK in tissue lysates treated with λPPase (B) or in pancreatic and WAT lysates prepared from mice injected with CHX (C). (D) Quantitation of percent of phosphorylated IRE1α in total IRE1α protein in various tissues shown in A. Values are mean ± SEM. Representatives of at least two independent experiments shown.
Figure 4
Figure 4. Fasting-refeeding induces mild ER stress in pancreas.
(A) Immunoblots of lysates from the pancreas of wildtype mice either fasted or fasted followed by 2 h refeeding (refed). For the PERK blot, a mixture of all 6 samples treated with CIP were included as a control. For the p-PERK blot, Tg-treated MEF cell lysates with or without CIP treatment were included as a control. HSP90, a loading control. (B) Quantitation of the percent of phosphorylated IRE1α in pancreas under fasting and refeeding conditions shown in A (N = 4 mice per cohort). (C) Q-PCR analyses of UPR genes in the pancreas under either fasting or refeeding. Values are mean ± SEM. Xbp1t, total Xbp1; Xbp1s/Xbp1t, splicing efficiency. N = 3–4 mice. *, P<0.05 using unpaired two-tailed Student's t-test. Representatives of at least two independent experiments shown.
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