Analysis of Total & pSer724 IRE1 alpha, the Sensor of ER Stress

Fri, 06/17/2016 - 13:01

Inositol-requiring protein 1/IRE1 alpha (also called Endoplasmic Reticulum to Nucleus Signaling 1/ERN1; predicted mol wt 110 kDa) is a serine-threonine protein kinase/endoribonuclease which plays a highly critical role in unfolded protein response/UPR signaling, a mechanism by which eukaryotic cells sense and deal with ER stress. The latter triggers growth arrest and apoptosis in cells with misfolded proteins. ER stress is initiated by three different ER-resident proteins: PERK (PRKR-like ER kinase), ATF-6 (activating transcription factor 6), and IRE1 Alpha, but the signaling mediated by IRE1 Alpha is the most conserved mechanism of UPR/ER stress in eukaryotes. IRE1 Alpha is expressed as a type-1 transmembrane protein in the ER and it has bifunctional cytosolic kinase as well as RNase domains. Under normal conditions, it is associated with the ER chaperone BiP /GRP78. During the sensing of misfolded proteins in the ER/UPR, IRE1 alpha gets dissociated which follows its di-or oligo-merization and trans-autophosphorylation through its kinase domain. The kinase activity of IRE1 Alpha is essential for the activation of its endoribonuclease domain. The activated IRE1 Alpha triggers its RNase activity towards Xbp1 mRNA which ultimately results in the production of active transcription factor XBP1. Activated XBP1 regulates the transcriptional expression of ER chaperone, lipogenic and endoplasmic reticulum associated degradation/ERAD genes (Walter and Ron 2011).

The transcriptional and enzymatic activities of IRE1 alpha are regulated through phosphorylation of Serine 724 residue (pSer-724IRE Alpha).  Phospho-IRE1 Alpha (Ser724) Antibodies are useful in the analysis of Serine 724 phosphorylation and several researchers have reported its immune-analysis using applications such as Western Blot (WB), Immunoprecipitation (IP), Immunocytochemistry/Immunofluorescence (ICC/IF), ELISA, and IHC-Paraffin/IHC-Frozen. Novus Biologicals is a leader in UPR research and Novus’ IRE1 alpha [p Ser724] Antibody NB100-2323 (over 120 citations) is the most referenced product in research publications documenting IRE1 Alpha mediated signaling. Novus offers total-IRE1 Alpha antibodies also which are useful for normalization of phosphor-IRE1 (Ser724) in WB densitometry or quantification of other immunoassays.

IRE1 alpha antibody


IRE1 alpha antibody

IRE1 alpha [pS724] Antibody [NB100-2323] 
WB detection of pS724 IRE1 alpha in lysates of Min6 cells which were treated with differential conc. of glucose for 3 hrs.


Total IRE1 alpha Antibody [NB100-2324] 
Western Blot detection of total Ire1 alpha protein in lysates from Min6 cells which were transfected with GFP-siRNA or Ire1-siRNA.

Also see
Phospho & Total IRE-1 Alpha Antibody Sampler (Catalog Number # NBP2-50067)

In a report documented in the Journal of Cellular Physiology, Vannuvel et al. 2016 employed Novus’ phospho-IRE1 alpha [S724] Antibody [NB100-2323] and Total IRE1 alpha Antibody [NB100-2324] for WB analysis of lysates from human hepatoma HepG2 cells which were subjected to ER stress induction with thapsigargin (TG, an inhibitor of the SERCA pumps) and brefeldin A (BFA, an inhibitor of the transport of proteins from the ER to the Golgi apparatus). TG was observed to cause a quicker and stronger induction of phosphorylated IRE1 Alpha in comparison to BFA-treatments. A recent study published in Biochemical and Biophysical Research Communications, Gao et al. 2016 reported the use of Novus’ IRE1 alpha [p Ser724] Antibody [NB100-2323] and other ER stress markers for the WB analysis of lysates from liver of control Sdit2+/+ and Sitd2 knockout (Sidt2−/−) mice. An enhanced expression of  UPR activation/ER stress markers (GRP78/BiP, CHOP, p-PERK, p-eIF2a, and p-IRE1a) was observed in livers from Sidt2−/− mice. The authors further suggested that ER stress might be implicated in the occurrence of nonalcoholic fatty liver disease (NAFLD) which was observed upon Sitd2 knockout in mice. Clarke et al. 2015 cited the use of Novus’ pSer724 IRE1 alpha [NB100-2323] and Total IRE1 alpha [NB100-2324] antibodies at 1:1000 dilution for the WB analysis of lysates from hypothalamus of mice which were subjected or not to 4 hour treatment of AD‐associated Abeta oligomers. This study established the effects of Abeta oligomers on brain hypothalamus and revealed molecular links between hypothalamic dysfunction in metabolic disorders and Alzheimer's disease/AD. Dr. Caigan Du’s team from University of British Columbia reported the use of antibodies for pIRE Alpha (Ser-724) and Total IRE1 alpha [NB100-2324] in WB analysis of lysates from Clusterin expressing renal tubular epithelial cell [TEC-CLU(hCLU)] and CLU-null TECs (TEC-CLU-/-) cells which were grown in K1+/+ medium and were exposed to normoxic (20% O2) or hypoxic (1% O2) conditions.  Total IRE1 expression was found to be induced by hypoxia in both TEC-CLU(hCLU) as well as TEC-CLU-/- cells, whereas, pIRE1 Alpha was enhanced in hypoxic TEC-CLUhCLU cells only. These findings signified the cytoprotective role of Clusterin (through pro-survival autophagy) in kidney tubular epithelial cells under hypoxic conditions (Alnasser et al. 2016).

Compiled by: Subhash Gangar


  1. Alnasser HA, Guan Q, Zhang F et al. 2016. Requirement of clusterin expression for prosurvival autophagy in hypoxic kidney tubular epithelial cells. Am J Physiol Renal Physiol.  310(2):F160-73 [PMID: 26561650].
  2. Clarke JR, Lyra E Silva NM, Figueiredo CP et al. 2015. Alzheimer-associated Aβ oligomers impact the central nervous system to induce peripheral metabolicderegulation. EMBO Mol Med. 7(2):190-210 [PMID: 25617315].
  3. Gao J, Zhang Y, Yu C et al. Spontaneous nonalcoholic fatty liver disease and ER stress in Sidt2 deficiency mice. Biochem Biophys Res Commun.  doi: 10.1016/j.bbrc.2016.05.122. [PMID: 27233614].
  4. Vannuvel K, Van Steenbrugge M, Demazy C et al. 2016. Effects of a Sublethal and Transient Stress of the Endoplasmic Reticulum on the Mitochondrial Population. J Cell Physiol. 231(9):1913-31 [PMID: 26680008].
  5. Walter P, Ron D. The unfolded protein response: from stress pathway to homeostatic regulation. Science. 2011;334:1081–1086. [PubMed 22116877].

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