In summary, we have optimized a sensitive and simple Phos-tag-based system to quantitatively assess ER stress and UPR activation with the following major advantages: First, dynamic ranges of PERK and IRE1α phosphorylation can be more sensitively visualized compared to regular SDS-PAGE gels; this is particularly important for physiological UPR where ER stress can be so mild that traditional methods may no longer be accurate or reliable. Second, the major breakthrough of our method lies in the unique pattern of IRE1α phosphorylation in the Phos-tag gel, which allows for a quantitative assessment of ER stress. To our knowledge, this is the first demonstration of quantitation of ER stress under physiological or pathological settings (e.g. the fasting-refeeding cycle or the accumulation of misfolded proteins). Finally, in comparison to using commercially-available phospho-specific antibodies (e.g. P-Ser724A IRE1α and P-Thr980 PERK), our method not only provides a complete view of the overall phosphorylation status of IRE1α and PERK proteins, but also circumvents the issue of whether these specific residues are indeed phosphorylated under certain physiological conditions.
Our data reveal that many tissues and cell types display constitutive basal UPR activity, presumably to counter misfolded proteins passing through the ER. This observation is in line with an early report demonstrating that under physiological conditions removal of these misfolded proteins in yeast requires coordinated action of UPR and ERAD 
. Taking it one step further, our data show that a fraction of mammalian IRE1α and PERK is constitutively active in many tissues, with ~10% IRE1α being phosphorylated and activated. This low level of IRE1α activation and ER stress in many tissues may provide a plausible explanation for the inability of an earlier study to detect basal UPR in the XBP1s-GFP reporter mice 
. We believe that this basal UPR activity, especially the IRE1α-XBP1 branch, is critical in maintaining ER homeostasis and providing quality control as supported by the embryonic lethality of IRE1α and XBP1-deficient mice 
. It is noteworthy that in skeletal muscles, IRE1α exhibited multiple non-phosphorylated bands while PERK protein is beyond the detection limit. As the IRE1α-XBP1 pathway is active in adult skeletal muscles 
, the role of UPR in myocytes is an interesting question as it may offer new insights into physiological UPR.
As exocrine pancreatic acinar cells account for over 80% of the pancreatic mass, pancreatic ER stress observed under the fasting-feeding cycle likely reflects the acute elevation of protein synthesis in acinar cells in response to food intake 
. Indeed, mice with XBP1 or PERK deficiency exhibit defective development of exocrine pancreas 
, suggesting an indispensable role for UPR in countering the fluctuating stress associated with food intake. While UPR is mildly active under fasting presumably to attenuate protein synthesis as previously suggested 
, our data showed a 3-fold increase of IRE1α phosphorylation, i.e. UPR, to enhance ER homeostasis in preparation for an upcoming wave of protein synthesis. Our results are in line with earlier observations demonstrating that ER in pancreatic acinar cells becomes dilated within 2–4 h refeeding 
. Nonetheless, it is quite surprising that ER stress in pancreatic cells fluctuates with the fasting-feeding cycle because acute mild UPR would expectedly reset proteostasis upon each fasting-feeding cycle, leading to the expansion of the proteostasis network and adaptation 
. Hence, we postulate that the proteostasis network in acinar cells is very flexible in order to respond to many variables in the feeding process. The same is likely to be true for pancreatic islet cells.
There are several potential applications for our method in both basic and clinical research. First, our method may help elucidate the activation mechanisms for IRE1α and PERK. The effect of critical residues or inter-/intra-molecular interactions on sensor activation as well as branch-specific activation of non-canonical UPR pathways can now be accurately measured and quantitated. Second, our method may aid in the diagnosis of UPR-associated diseases by providing a more sensitive tool for detecting ER stress. The knowledge of the extent of ER stress in a given tissue of a patient may help assess disease progression. Finally, our method may assist in drug development and design. The efficacy of drugs such as chemical chaperones or antioxidants on ER stress can be quantitatively measured based on sensor activation, circumventing the complications associated with crosstalk among various pathways.
As ER stress is being implicated in an increasing number of physiological processes as well as human diseases such as cancer, liver diseases, neurodegeneration and type-1 diabetes 
, new strategies and approaches enabling a comprehensive understanding of UPR in physiological and disease settings are urgently needed to facilitate drug design targeting UPR in conformational diseases 
. The ability to directly visualize and quantitate UPR activation is an important step towards gaining novel insights into physiological UPR and improving therapeutic strategies targeting UPR in vivo.