Ire1α

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, Canada, China

IRE1α is a protein that functions as a sensor of endoplasmic reticulum (ER) stress. It plays a key role in the unfolded protein response (UPR), a cellular signaling pathway that is activated in response to the accumulation of unfolded or misfolded proteins in the ER.

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166 protocols using ire1α

Western Blot Analysis of ER Stress Markers

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We lysed the cells in RIPA buffer (Sigma Aldrich) containing 150 mM NaCl, 1.0% Nonidet-P 40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tri (pH 8.0), protease inhibitor cocktail, and PhoSTOP (Roche Molecular Biochemicals, Basel, Switzerland) as described previously^{17 (link)}. We used the following primary antibodies: PPARγ, C/EBPα, CHOP, GRP78, PERK, p-PERK, p-eIF2α, eIF2α, IRE1α, IRE1α, FABP4, perilipin, and α-tubulin (1:1000; Cell Signaling Technology, Danvers, MA, USA).
Secondary antibodies (anti-rabbit IgG or anti-mouse IgG, 1:2000)) were purchased from Cell Signaling Technology. We conducted immunoreactive detection of specific proteins using an ECL Western blotting kit (GE, Hercules, CA, USA) according to the manufacturer’s instructions.

Kang S.U., Kim H.J., Kim D.H., Han C.H., Lee Y.S, & Kim C.H. (2018). Nonthermal plasma treated solution inhibits adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes via ER stress signal suppression. Scientific Reports, 8, 2277.

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Western Blot Analysis of Esophageal Tissue

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Frozen rat esophagus tissue was homogenized in Tissue Protein Extraction Reagent (ThermoFisher, Waltham, MA, USA) using TissueLyser II (Qiagen) at 30 Hz for 5 min. Extracted protein was then quantified using the DC Protein Assay (Bio-Rad). Western blot analysis was performed as previously described [123 (link)]. Images were captured using the Bio-Rad ChemiDoc Imaging System and quantified by means of chemiluminescent immunodetection using Bio-Rad Image Lab Software version 6.1.0 with expression levels normalized to the loading control GAPDH. Immunoblotting was performed using commercially available antibodies from Abcam (Cambridge, MA, USA) and Cell Signaling Technology (Danvers, MA, USA): ABST (ab203205, 1:500), ATF-4 (CST #11815, 1:1000), CD44 (ab189524, 1:500), GAPDH (CST #2118, 1:25,000), HSP60 (CST #12165, 1:5000), IRE1α (CST #3294, 1:1000), and phospho-AMPK (CST #2535, 1:1000). Patient EAC samples with matched normal and BE tissues were collected at the University Hospital at the University of Michigan. Informed consent was obtained from patients prior to sample collection. Protein extraction and quantification were similarly performed as described above.

Zhang Y., Weh K.M., Tripp B.A., Clarke J.L., Howard C.L., Sunilkumar S., Howell A.B, & Kresty L.A. (2023). Cranberry Proanthocyanidins Mitigate Reflux-Induced Transporter Dysregulation in an Esophageal Adenocarcinoma Model. Pharmaceuticals, 16(12), 1697.

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Protein Expression Analysis in Mouse Tissues

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The total protein of mouse breast tissues and MAC-T cells was separated by RIPA reagent (Biosharp, China). The BCA Protein Assay Kit (Thermo Scientific, MA, USA) was performed to detect protein concentration. The protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); then, polyvinylidene difluoride (PVDF) membranes were used to receive the transferred protein from the gel. The membranes were blocked with 5% skimmed milk for 2 h; then, the primary antibodies (BiP, Ero1-Lα, PDI, IRE1α, PERK, CHOP, JNK, ERK, p38, p65, and β-actin from Cell Signaling Technology, USA; PPARγ from Abcam, UK) were incubated at 4°C overnight, and then, the secondary antibody (anti-rabbit IgG and HRP-linked antibody from Cell Signaling Technology, USA) was incubated at 37°C for 1 h. Finally, the immunoblot signal was displayed with ECL ultrasensitive chemiluminescent solution with chemiluminescence imaging system.

Chen Y., Yang J., Huang Z., Yin B., Umar T., Yang C., Zhang X., Jing H., Guo S., Guo M., Deng G, & Qiu C. (2022). Vitexin Mitigates Staphylococcus aureus-Induced Mastitis via Regulation of ROS/ER Stress/NF-κB/MAPK Pathway. Oxidative Medicine and Cellular Longevity, 2022, 7977433.

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Western Blot Analysis of Stress Signaling

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Proteins were extracted with cell lysis buffer containing 1% Triton X-100, sonicated using QSonica-Q800R2, quantified using bicinchoninic acid assay (Sigma) and separated on NuPAGE™ 4-12% Bis-Tris precast gels (Thermo Fisher). Immunoblot analyses were conducted according to standard protocol with the following antisera: caspase-3 (#9662), PARP (#9542), β-actin (#3770), IRE1α (#3294), p-eIF2α (#3597), t-eIF2α (#2103), p-P38 MAPK (#4511), t-P38 MAPK (#8690), p-JNK1/2 (#4668), t-JNK1/2 (#9252), p-ERK1/2 (#9101), t-ERK1/2 (#9102), CHOP (#5554), Bim (#2933), Bcl-2 (#4223), Bax (#5023), p-Akt (#4060), t-Akt (#9272), GAPDH (#5174), cyclin b1 (#12231), p-CDK1 substrates (#9477) and FLAG (#14793) from Cell Signaling Technology; p62 (#ab56416) and Mcl-1 (#ab31948) antibodies were purchased from Abcam, while LC3 antibody (#NB100-2220) was obtained from Novus Biologicals. Images were captured using a ChemiDoc™ Touch Imaging System and processed using ImageLab™ Software.

Trapika I.G., Liu X.T., Chung L.H., Lai F., Xie C., Zhao Y., Cui S., Chen J., Tran C., Wang Q., Zhang S., Don A.S., Li G.Q., Hanrahan J.R, & Qi Y. (2021). Ceramide Regulates Anti-Tumor Mechanisms of Erianin in Androgen-Sensitive and Castration-Resistant Prostate Cancers. Frontiers in Oncology, 11, 738078.

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Dissecting Apoptosis Signaling Pathways

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G1 was purchased from Tocris (Wiesbaden-Nordenstadt, Germany), dissolved (5 mM) in dimethyl sulfoxide (DMSO) (Roth, Karsruhe, Germany) and stored at −20 °C; Thapsigargin, SP600125, GSK2606414 were also purchased from Tocris. Indo-1 AM was from Thermo Fisher Scientific (Waltham, MA, USA). zVAD-fmk was bought from Santa Cruz (Santa Cruz, CA, USA). SB203580 and Kira6 were purchased from MERCK Millipore (Darmstadt, Germany). All substances were dissolved in DMSO. Antibodies were obtained from the following commercial sources: caspase 9 (Ca# 9502), cleaved PARP (Ca# 9541), IRE1α (Cat# 3294), PERK (Cat# 3192), eIF2α (Cat# 5234), phospho-eIF2α (Cat# 3398), BiP GRP78 (Ca# 3177), CHOP (Cat# 2895), p38 MAPK (Ca# 9212), phospho-p38 MAPK (Ca# 4511), phospho-SAPK/JNK (Ca# 4668), caspase 3 (Ca# 9662), BCL-2 (Ca# 2872), Cell Signaling (Danvers, MA, USA); ATF6 (Cat# 73500), BioAcademia (Osaka, Japan); puromycin (Ca# MABE343), cylophilin D (Ca# AP1035), MERCK Millipore (Darmstadt, Germany); phospho-IRE1α (Cat# NBP2-50067), Novus Biologicals (Littleton, CO, USA); cytochrome c (Ca# 556433), BD Biosciences (Franklin Lakes, NJ, USA); β-actin (Cat# A5441, Sigma-Aldrich (Steinheim, Germany)). Secondary, peroxidase-conjugated antibodies were purchased from Dianova (Hamburg, Germany). All other chemicals of analytical grade were obtained from Sigma-Aldrich or Roth.

Vo D.K., Hartig R., Weinert S., Haybaeck J, & Nass N. (2019). G-Protein-Coupled Estrogen Receptor (GPER)-Specific Agonist G1 Induces ER Stress Leading to Cell Death in MCF-7 Cells. Biomolecules, 9(9), 503.

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Western Blot Analysis of ER Stress Markers

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For Western blot analysis, a 4–20% Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad Laboratories) was used. Each sample was then denatured at 94°C for 3 min before being loaded. Gels were blotted onto TransBlot Turbo polyvinylidene fluoride membranes (Bio-Rad Laboratories), and blocked in PBST buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, and 0.1% Tween 20, pH 7.42) containing 5% nonfat dry milk. The signal was measured by chemoluminescence (ECL plus; GE Healthcare). Western blots were performed using rabbit anti-mouse HERP (Santa Cruz Biotechnology, Inc.), CHOP, BIP, IRE1α (Cell Signaling Technology), goat anti–mouse Igκ (Beckman Coulter), and using goat anti–mouse GAPDH (R&D Systems) Abs for normalization.

Srour N., Chemin G., Tinguely A., Ashi M.O., Oruc Z., Péron S., Sirac C., Cogné M, & Delpy L. (2016). A plasma cell differentiation quality control ablates B cell clones with biallelic Ig rearrangements and truncated Ig production. The Journal of Experimental Medicine, 213(1), 109-122.

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Western Blot Analysis of NAFLD Markers

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Total cell lysates were extracted from HepG2 cells using cell lysis buffer (PH 6.8, 50 mmol/L Tris, 1% SDS, protease inhibitor). Nuclear protein was extracted using NucBuster™ Protein Extraction Kit (71183‐3, Novagen). Protein concentration was detected by BCA method. Boiled cell lysates were subjected to SDS‐PAGE separation on 8%‐12% gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). Primary antibodies against PNPLA3 (ab81874, Abcam), NF‐kB p65 (ab4764, Abcam), SREBP‐1 (ab3259, Abcam), SP3 (sc‐644, Santa Cruz), TNF‐α (3707, Cell Signaling Technology), Bip (3183, Cell Signaling Technology), c‐Jun (9165, Cell Signaling Technology), IRE‐1α (3294, Cell Signaling Technology), JNK2 (9258, Cell Signaling Technology), pJNK (Cell Signaling Technology), or β‐actin (4970, Cell Signaling Technology) were purchased commercially. Antimouse or anti‐rabbit fluorescent secondary antibodies were used, and the bands were visualized using the Odyssey infrared fluorescence imaging system (LI‐COR). The arachidonic acid (AA) levels in cell culture supernatants were measured using an ELISA kit (OKEH02583, Aviva Systems Biology) according to the manufacturer's instructions.

Yuan S., Liu H., Yuan D., Xu J., Chen Y., Xu X., Xu F, & Liang H. (2019). PNPLA3 I148M mediates the regulatory effect of NF‐kB on inflammation in PA‐treated HepG2 cells. Journal of Cellular and Molecular Medicine, 24(2), 1541-1552.

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Osthole Signaling Pathway Regulation

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Osthole (catalog number: O9265) was purchased from Sigma (St. Louis, MO, USA). Osthole was dissolved in DMSO to prepare a chemical stock for treatment. Antibodies against phosphorylated Akt (Ser⁴⁷³, catalog number: 4060), P70S6K (Thr⁴²¹/Ser⁴²⁴, catalog number: 9204), S6 (Ser²³⁵/Ser²³⁶, catalog number: 2211), ERK1/2 (Thr²⁰²/Tyr²⁰⁴, catalog number: 9101), p90RSK (Thr⁵⁷³, catalog number: 9346), JNK (Thr¹⁸³/Tyr¹⁸⁵, catalog number: 4668), total Akt (catalog number: 9272), P70S6K (catalog number: 9202), S6 (catalog number: 2217), ERK1/2 (catalog number: 4695), p90RSK (catalog number: 9335), JNK (catalog number: 9252), IRE1α (catalog number: 3294), eIF2α (catalog number: 5324), Bak (catalog number: 12105S), and Bax (catalog number: 2772) were purchased from Cell Signaling Technology (Beverly, MA, USA). Bcl-xL, p-Bcl-2, cleaved caspase 3 and cleaved caspase 9 were also purchased from cell Signaling Technology. Antibodies against GRP78 (catalog number: sc-13968), ATF6 α (catalog number: sc-166659), and α-tubulin (TUBA, catalog number: sc-32293) were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Inhibitors of ERK1/2 (U0126, catalog number: E1282) and JNK (SP600125, catalog number: E1305) were purchased from Enzo Life Sciences, Inc (Farmingdale, NY, USA), and a PI3K/Akt inhibitor (LY294002, catalog number: 9901) was purchased from Cell Signaling Technology, Inc.

Park W., Park S., Song G, & Lim W. (2019). Inhibitory Effects of Osthole on Human Breast Cancer Cell Progression via Induction of Cell Cycle Arrest, Mitochondrial Dysfunction, and ER Stress. Nutrients, 11(11), 2777.

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Antibodies and Reagents for ER Stress Study

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Primary antibodies targeting the following proteins were used in this study: p-eIF2α, IRE-1α, CHOP (Cell Signaling Technologies, Danvers, MA, USA), GRP78, ATF6α, PERK, p-PERK, sXBP-1, β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), p-IRE-1α (Abcam, Cambridge, MA, USA) and monoclonal PDI (clone 1D3, Enzo Life Sciences, Farmingdale, NY, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology. IC87114 was obtained from Calbiochem (San Diego, CA, USA). Ovalbumin, lipopolysaccharide, N-ethylmaleimide (NEM), 4-hydroxy-2-nonenal (4-HNE) and dihydroethidine (DHE) were purchased from Sigma-Aldrich (St Louis, MO, USA).

Kim H.K., Lee G.H., Bhattarai K.R., Junjappa R.P., Lee H.Y., Handigund M., Marahatta A., Bhandary B., Baek I.H., Pyo J.S., Kim H.K., Chai O.H., Kim H.R., Lee Y.C, & Chae H.J. (2018). PI3Kδ contributes to ER stress-associated asthma through ER-redox disturbances: the involvement of the RIDD–RIG-I–NF-κB axis. Experimental & Molecular Medicine, 50(2), e444-.

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Western Blot Analysis of Cardiac Proteins

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Protein extracts from frozen mouse, rat, and human hearts, AMVMs and NRVMs were prepared by lysis in ice-cold modified RIPA buffer (150 mM NaCl, 50 mM Tris HCL pH 7.4, 1% Triton-X 100, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 2 mM EDTA) containing protease and phosphatase inhibitors. Proteins were separated by SDS-PAGE on 4%−20% gradient gels (Bio-Rad) and transferred to nitrocellulose membranes. An Odyssey scanner (LI-COR version 3.0) was used as detection system. For human myocardial samples, protein lysates were prepared using cell lysis buffer (CST, Cell Signaling Technology) with the addition of 1mM phenylmethylsulfonyl fluoride (PMSF). Lysates were prepared using the Retsch Mixer Mill. Capillary-based immunoassay was performed using the Wes-Simple Western method with the anti-rabbit detection module (ProteinSimple). Protein expression was measured by chemiluminescence and quantified as area under the curve using the Compass for Simple Western program (ProteinSimple). Proteins were detected with the following primary antibodies: pIRE1α [Ser724] (NB100–2323, Novus Biological); IRE1α (#3294, Cell Signaling), GRP78 (#3177, Cell Signaling); anti-GRP94 (#2104, Cell Signaling); PERK (#3192, Cell Signaling); ATF6 (24169–1-AP, Proteintech); iNOS (#13120, Cell Signaling); nNOS (#4234, Cell Signaling); eNOS (#9572, Cell Signaling); GAPDH (10R-G109a, Fitzgerald).

Schiattarella G.G., Altamirano F., Tong D., French K.M., Villalobos E., Kim S.Y., Luo X., Jiang N., May H.I., Wang Z.V., Hill T.M., Mammen P.P., Huang J., Lee D.I., Hahn V., Sharma K., Kass D.A., Lavandero S., Gillette T.G, & Hill J.A. (2019). Nitrosative Stress Drives Heart Failure with Preserved Ejection Fraction. Nature, 568(7752), 351-356.

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