Phospho-ERN1 (S724) Recombinant Monoclonal Antibody
Description
Mechanistic Role of ERN1 S724 Phosphorylation
Phosphorylation at S724 is a hallmark of ERN1 activation. This site lies within the kinase activation loop, enabling autophosphorylation and subsequent activation of its endoribonuclease domain. Key functions include:
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UPR Activation: Triggers splicing of XBP1 mRNA, producing a transcription factor that upregulates stress-response genes .
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ER Stress Signaling: Critical for resolving protein misfolding in the ER via XBP1-dependent pathways .
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Cross-Species Conservation: S724 phosphorylation is conserved in human, mouse, and rat models .
Western Blot (WB)
The antibody is widely used to detect ERN1 phosphorylation in cellular lysates under ER stress.
Key Insight: In Ern1 S724A/S724A mice, phosphorylation at S724 is undetectable, confirming the antibody’s specificity .
Immunofluorescence (IF)
Used to localize ERN1 in stressed cells.
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Protocol: Fixed HeLa cells (4% formaldehyde), permeabilized (0.2% Triton X-100), blocked (10% goat serum), and incubated with antibody overnight .
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Outcome: Cytoplasmic ERN1 signal with DAPI counterstaining .
Immunohistochemistry (IHC)
Validated in paraffin-embedded human spleen tissue (1:300 dilution) with hematoxylin counterstaining .
Comparative Analysis with Other Antibodies
The recombinant monoclonal variant (e.g., CSB-RA007795A724phHU) outperforms traditional polyclonal antibodies in specificity and lot consistency.
Role in ER Stress-Induced Pathways
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XBP1 Splicing: S724 phosphorylation is essential for IRE1α’s RNase activity. In Ern1 S724A/S724A mice, XBP1 splicing is blunted under tunicamycin-induced stress, leading to exacerbated hepatic steatosis .
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RIDD Activity: While S724 phosphorylation primarily drives XBP1 splicing, its absence may shift IRE1α toward regulated IRE1-dependent decay (RIDD) of other mRNAs .
Disease Relevance
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Cancer and Metabolic Disorders: ERN1 S724 phosphorylation is implicated in tumor adaptation to hypoxia and metabolic stress .
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Neurodegeneration: Dysregulated UPR pathways, including IRE1α activation, are linked to neurodegenerative diseases .
Technical Considerations
Product Specs
The Phospho-ERN1 (S724) Recombinant Monoclonal Antibody is generated by cloning the coding sequence for the phospho-ERN1 (S724) monoclonal antibody (produced by immunizing animals with the synthetic phosphopeptide of ERN1) into plasmids and transfecting the clones into cell lines. This antibody is a rabbit IgG purified using affinity chromatography. The Phospho-ERN1 (S724) antibody specifically detects endogenous levels of human ERN1 only when phosphorylated at Ser724. This antibody can be used in ELISA, Western Blot (WB), and Immunofluorescence (IF) analyses.
Phosphorylation at Ser724 residue of the ERN1 protein is crucial for regulating its transcriptional and enzymatic activities. Phospho-ERN1 (S724) antibodies are valuable tools to analyze ERN1 Ser724 phosphorylation, a highly conserved site within the kinase activation domain.
Target Background
ERN1 (also known as IRE1α) is a serine/threonine-protein kinase and endoribonuclease that plays a critical role as a sensor for the endoplasmic reticulum unfolded protein response (UPR). In unstressed cells, the endoplasmic reticulum luminal domain of ERN1 is maintained in its inactive monomeric state by binding to the endoplasmic reticulum chaperone HSPA5/BiP. The accumulation of misfolded proteins in the endoplasmic reticulum leads to the release of HSPA5/BiP, allowing the luminal domain to homodimerize, triggering autophosphorylation of the kinase domain and subsequent activation of the endoribonuclease activity.
The endoribonuclease activity of ERN1 is specific for XBP1 mRNA, excising 26 nucleotides from it. This splicing event generates a spliced transcript of XBP1, encoding a transcriptional activator protein that upregulates expression of UPR target genes. ERN1 also acts as an upstream signal for ER stress-induced GORASP2-mediated unconventional (ER/Golgi-independent) trafficking of CFTR to the cell membrane by modulating the expression and localization of SEC16A.
- Research suggests that apocynin protects endothelial cells against ER stress-induced apoptosis by involving IRE1alpha. This finding may provide a novel mechanistic explanation for the anti-apoptotic effect of apocynin in ER stress. PMID: 29696609
- Studies have revealed that DDRGK1 is essential for regulating endoplasmic reticulum (ER) homeostasis in both human cancer cells and mouse hematopoietic stem cells. Depletion of DDRGK1 activates the apoptotic pathway by targeting the ER-stress sensor IRE1alpha. DDRGK1 regulates IRE1alpha protein stability through its interaction with the kinase domain of IRE1alpha. PMID: 28128204
- IRE1 was upregulated during excisional wound healing at a time consistent with the proliferative phase of wound healing. Inhibition of IRE1 led to decreased scar formation. PMID: 29316036
- Urinary levels of the spliced X-box binding protein 1 (sXBP1) mRNA are used as a proxy for inositol-requiring enzyme 1alpha (IRE1alpha) activity, as sXBP1 is absolutely sensitive and specific for endoplasmic reticulum stress. PMID: 29276149
- This study demonstrated that, similar to yeast, human IRE1alpha's endoplasmic reticulum-lumenal domain (hIRE1alpha LD) binds peptides with a characteristic amino acid bias. Peptides and unfolded proteins bind to hIRE1alpha LD's MHC-like groove, inducing allosteric changes that lead to its oligomerization. PMID: 28971800
- Three branches of the Unfolded Protein Response (UPR) have been identified, including the activation of the inositol-requiring enzyme 1 (IRE1), the pancreatic ER kinase (PKR)-like ER kinase (PERK), and the activating transcription factor 6 (ATF6). PMID: 28105371
- IRE1alpha is expressed at lower levels in higher-grade gliomas, suggesting a greater antitumor efficacy of the oncolytic virus M1. These findings highlight a defensive mechanism of glioma cells against the oncolytic virus M1 and identify possible approaches to enhance the oncolytic viral protein accumulation and subsequent lysis of tumor cells. PMID: 29263275
- Systematic mutation of the AREs (ARE1-3) in the LDLR 3'UTR and expression of each mutant coupled to a luciferase reporter in Huh7 cells demonstrated that ARE1 is required for rapid LDLR mRNA decay and 5-AzaC-induced mRNA stabilization via the IRE1alpha-EGFR-ERK1/2 signaling cascade. PMID: 29208426
- Pharmacologic modulation of IRE1 counteracts metaflammation and alleviates atherosclerosis. PMID: 28137856
- This study demonstrated that fine-tuning of the expression of proliferation-related transcription factor genes depends on glucose and glutamine deprivation in an IRE1-dependent manner and possibly contributes to slower tumor growth after inhibition of IRE1. PMID: 29537195
- The activation of IRE1alpha by the hepatitis C virus protein NS4B in XBP1-proficient cells conferred apoptosis resistance and promoted viral replication. PMID: 28588082
- This study highlighted an important role for Nck1 in fine-tuning IRE1alpha expression and signaling that regulate PTP1B expression and subsequent activation of the PI3K-Akt pathway in HepG2 cells. PMID: 28455143
- IRE1alpha-XBP1 pathway regulates Mel-RMu cell proliferation and progression by activating IL-6/STAT3 signaling. PMID: 28222747
- The authors propose that the Sec61-IRE1alpha complex defines the extent of IRE1alpha activity and may determine cell fate decisions during endoplasmic reticulum stress conditions. PMID: 28504640
- ERN1 and ALPK1 inhibit differentiation of bi-potential tumor-initiating cells in human triple-negative breast cancer. PMID: 27829216
- IRE1 deficiency fully restored the learning and memory capacity of Alzheimer's disease (AD) mice, associated with improved synaptic function and enhanced long-term potentiation (LTP). At the molecular level, IRE1 deletion reduced the expression of amyloid precursor protein (APP) in cortical and hippocampal areas of AD mice. PMID: 28341998
- The biologic processes altered by aberrant IRE1alpha-XBP1 signaling in these innate immune cells. PMID: 26979393
- Inhibition of IRE1 modifies the hypoxic regulation of GADD34 family gene expression in cultured glioma cells. PMID: 29227599
- Changes in the expression level of nuclear genes encoding mitochondrial proteins possibly reflect metabolic reprogramming of mitochondria by hypoxia and IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation under inhibition of the IRE1 enzyme function. PMID: 29235326
- The inhibition of IRE1 changes sensitivity of the expression of down-stream genes to glutamine deprivation in cultured glial tumor cells. PMID: 29235329
- The present study demonstrated that the inhibition of IRE1 in glioma cells affected the hypoxic regulation of the expression of down-stream genes in various directions, although hypoxic conditions did not abolish the effect of IRE1 inhibition on the expression of respective genes. PMID: 29235836
- Inhibition of IRE1 modifies hypoxic regulation of pentose-phosphate pathway genes expression in cultured glioma cells. PMID: 29236388
- This study reports that the endoplasmic reticulum luminal co-chaperone ERdj4/DNAJB9 is a selective IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1alpha. PMID: 29198525
- The present study is the first to uncover a key prosurvival modulator, Yip1A, which coordinates IRE1 signaling with PERK signaling to support the survival of HeLa and CaSki cervical cancer cells. PMID: 28358375
- Inositol-requiring kinase 1 may be a useful biomarker to predict recurrence in surgically resected lung adenocarcinoma patients. PMID: 28334878
- Fortilin directly interacts with the cytoplasmic domain of IRE1alpha, inhibits both kinase and endoribonuclease (RNase) activities of this stress sensor, and protects cells against apoptotic cell death at both cellular and whole animal levels. PMID: 28550308
- Overall, these data demonstrate that hypoxia can suppress adiponectin expression and activate the PERK and IRE1 signaling pathways in differentiated adipocytes, and these two pathways are involved in the suppression of adiponectin expression induced by hypoxia. PMID: 28888981
- ER stress-regulated IRE1-dependent decay is involved in regulating hepatic diseases. (review) PMID: 27774654
- The unfolded protein response reduces glucose metabolism via IRE1 signaling. PMID: 28093214
- Results of this investigation demonstrate that inhibition of IRE1 signaling enzyme function affects the expression of NRIP1, EBBP, ESRRA, E2IG5, PGRMC2, and SLC39A6 genes in U87 glioma cells in a gene-specific manner, and these changes possibly contribute to the suppression of cell proliferation. Most of these genes are regulated by hypoxia and preferentially through the IRE1 signaling pathway of endoplasmic reticulum stress. PMID: 28222026
- IRE1alpha was shown to cleave miR-150, thereby releasing the suppressive effect that miR-150 exerted on alphaSMA expression through c-Myb. Inhibition of IRE1alpha was also demonstrated to block endoplasmic reticulum expansion through an XBP-1-dependent pathway. PMID: 27226027
- IRE-1 has an ancient function as a cytoplasmic sentinel that activates p38 and SKN-1(Nrf2). Cysteine modifications induced by ROS signals can direct proteins to adopt unexpected functions and may coordinate many cellular processes. PMID: 27540856
- The findings indicate that IRE1-XBP1 downregulation distinguishes germinal center B-cell-like diffuse large B-cell lymphoma (DLBCL) from other DLBCL subtypes and contributes to tumor growth. PMID: 28167662
- Western blot analysis of subcutaneously implanted AsPC-1 and BxPC-3 tumors as well as orthotopically implanted Panc-1 tumors demonstrated upregulation of BIP, CHOP, and IRE1alpha expression in the tumor lysates from penfluridol-treated mice compared to tumors from control mice. PMID: 28618969
- The ABL family of tyrosine kinases rheostatically enhances IRE1alpha's enzymatic activities, thereby potentiating endoplasmic reticulum stress-induced apoptosis. PMID: 28380378
- Pre-ischemia melatonin treatment alleviated acute neuronal injury after ischemic stroke by inhibiting endoplasmic reticulum stress-dependent autophagy via PERK and IRE1 signaling pathways. PMID: 28178380
- IRE1 is involved in multivesicular body information during endoplasmic reticulum stress. PMID: 27725157
- Comparison of this structure with other existing structures of IRE1alpha and integration of our extensive structure activity relationship (SAR) data has led us to formulate a model to rationalize how ATP-binding site ligands are able to control the IRE1alpha oligomeric state and subsequent RNase domain activity. PMID: 27227314
- Our data indicate that reduced response of the IRE1alpha/Xbp-1 signaling pathway to bortezomib may contribute to drug resistance in myeloma cells. PMID: 27647225
- Heat stress simultaneously activates both the unfolded protein response (UPR) and autophagy, followed by the activation of a negative feedback system in UPR by modulating the responses related to the IRE1alpha-XBP-1 axis. PMID: 27743894
- These findings underscore the essential role of cytosine nucleotide at +1 in the 3' splice site for determining cleavage specificity of hIRE1alpha. PMID: 28027394
- Crucially, Chlamydia trachomatis infection resulted in robust IRE1alpha RNAse activity that was dependent on TLR4 signaling, and inhibition of IRE1alpha RNAse activity prevented PKR activation. PMID: 27021640
- Pretreatment by IRE1 agonist tunicamycin or JNK agonist anisomycin attenuated the effect of psoralen on osteoporotic osteoblasts. Psoralen inhibited apoptosis of osteoporotic osteoblasts by regulating the IRE1-ASK1-JNK pathway. PMID: 28349059
- Results indicate that excessive activation of the endoplasmic reticulum stress-associated IRE1alpha pathway is involved in LC neuronal apoptosis induced by single prolonged stress exposure, which may be a crucial mechanism in the pathogenesis of post-traumatic stress disorder. PMID: 27059130
- These results confirmed that ER stress-mediated apoptosis contributes to the protective effects of naringenin against H/R injury, potentially involved in ATF6, IRE1alpha, and PERK signaling activation. PMID: 27785700
- Structural and mechanistic studies of IRE1. PMID: 27686654
- Inhibition of IRE1 signaling enzyme function affects the expression of NR3C1 and related genes in U87 glioma cells in a gene-specific manner, and all these genes are regulated by hypoxia preferentially through the IRE1 signaling pathway of endoplasmic reticulum stress. PMID: 27560795
- Data suggest that ubiquitin D (UBD) provides a negative feedback on cytokine-induced activation of the endoplasmic reticulum to nucleus signaling 1 (IRE1alpha)/c-Jun N-terminal kinase (JNK) pro-apoptotic pathway in cytokine-exposed beta cells. PMID: 27044747
- We used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1alpha (IRE1alpha)/X-box binding protein-1. PMID: 26621917
- Using drugs that specifically inhibit or activate the PERK or IRE1alpha sensors, we demonstrate that signaling through the PERK axis activates this expression, through a transcriptional mechanism. PMID: 26634309
Customer Reviews
Applications : Immunoblot analysis
Sample type: cell
Review: Immunoblot analysis of IRE1α in lysates of Kupffer cells and hepatocytes freshly isolated from livers of male MΦKO mice or Ern1-flox/flox control littermates. HSP90 was used as the loading control. B–E, MΦKO or floxed control mice were subjected to sham or hepatic warm ischemia/reperfusion (n=3–4 per group).
