Isrib

Manufactured by Cayman Chemical

Sourced in United States

ISRIB is a chemical compound that inhibits the integrated stress response (ISR) pathway. It functions by blocking the phosphorylation of the eukaryotic translation initiation factor 2 subunit alpha (eIF2α), which is a key step in the ISR activation. This product is intended for research use only.

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Lab products found in correlation

Thapsigargin, by Fujifilm (2 mentions) Gsk2656157, by Cayman Chemical (2 mentions) Mss205172, by Thermo Fisher Scientific (1 mentions) Mss205173, by Thermo Fisher Scientific (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Stealth rnai, by Thermo Fisher Scientific (1 mentions) Anti phospho eif2α, by Cell Signaling Technology (1 mentions) Anti β actin, by Merck Group (1 mentions) Anti p erk, by Cell Signaling Technology (1 mentions) Cycloheximide (chx), by Fujifilm (1 mentions) Anti puromycin, by Merck Group (1 mentions) Gcn2ib, by MedChemExpress (1 mentions) Anti g3bp1, by BD (1 mentions) Anti phospho gcn2, by Abcam (1 mentions) Gsk2606414, by Cayman Chemical (1 mentions) Gsk2606414, by Fujifilm (1 mentions) Anti gcn2, by Cell Signaling Technology (1 mentions) Anti tia1, by Proteintech (1 mentions) Anti atf4, by Proteintech (1 mentions) Anti tdp 43, by Proteintech (1 mentions)

Spelling variants of this product

Trans-ISRIB (7 citations)

7 protocols using isrib

Myogenic Differentiation in C2C12 Cells

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A mouse myoblast cell line, C2C12, was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37°C under 5% CO2.
Myogenic differentiation was induced by replacing the medium with the differentiation medium, DMEM supplemented with 2% horse serum. C2C12 myoblasts were transfected with 50 nM of Stealth RNAi (Thermo Fisher Scientific, Waltham, MA, USA) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's protocol.
The following siRNAs were used: Stealth RNAi siRNA negative control (Negative Control, Med GC, Thermo Fisher Scientific), Stealth RNAi for Myoparr, and Stealth RNAi siRNAs specific for hnRNPK (MSS205172 and MSS205173, Thermo Fisher Scientific). The siRNA sequences are listed in Supplemental Table 1. At 24 h after siRNA transfection, myogenic differentiation was induced. At 24 h or 72 h after differentiation induction, cells were collected for the analysis of RNAs and proteins. For ISRIB treatment, the differentiation medium was added either with or without 1 µM ISRIB (Cayman Chemical Company, Ann Arbor, MI, USA).

Hitachi K., Kiyofuji Y., Nakatani M., & Tsuchida K. (2021). Myoparr-associated and -independent multiple roles of heterogeneous nuclear ribonucleoprotein K during skeletal muscle cell differentiation.

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Cellular stress response regulation assay

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24S-OHC [52 (link)] was dissolved in EtOH (Wako, Osaka, Japan). F12511 was the generous gift of Kowa (Aichi, Japan). Thapsigargin and CHX were purchased from Wako (Osaka, Japan). GSK2606414 and ISRIB were from Cayman Chemical (Ann Arbor, MI, USA). GCN2iB was from MedChemExpress (Monmouth Junction, NJ, USA). Thapsigargin, GSK2606414, CHX, and ISRIB were dissolved in dimethyl sulfoxide (DMSO; Wako). The following antibodies were from commercial sources: anti-PERK (Cat# 3192), anti-phospho-eIF2α (Cat# 3398), anti-eIF2α (Cat# 5324), and anti-GCN2 (Cat# 3302) were from Cell Signaling (Danvers, MA, USA); anti-β-actin (Cat# A5441) was from Sigma-Aldrich (St. Louis, MO, USA); anti-TIA1 (Cat# 12133-2-AP), anti-ATF4 (Cat# 10835-1-AP), and anti-TDP-43 (Cat# 12782-2-AP) were all from Proteintech (Chicago, IL, USA); anti-G3BP1 (Cat# 611126) was from BD Biosciences (Franklin Lakes, NJ, USA); anti-phospho-GCN2 (Cat# ab75836) was from Abcam (Cambridge, UK); and anti-puromycin (Cat# MABE343) was from Merck Millipore (Burlington, MA, USA); All other chemicals, of analytical grade, were obtained from Sigma-Aldrich or Wako.

Urano Y., Osaki S., Chiba R, & Noguchi N. (2022). Integrated stress response is involved in the 24(S)-hydroxycholesterol-induced unconventional cell death mechanism. Cell Death Discovery, 8, 406.

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Intravitreal ISRIB Injection Protocol

Cited 1 time

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The intravitreal injections were performed as described [51 (link)] with the following modifications. Immediately after the laser coagulation, 9.0 pg or 9.0 ng of ISRIB (Cayman Chemical, Ann Arbor, MI, USA) in 2 µL [10 nM or 10 µM, dissolved in phosphate-buffered saline (PBS) containing 0.1% dimethyl sulfoxide (DMSO)] was injected into the vitreous cavity with a Hamilton glass syringe (701N; Hamilton Co., Reno, NV, USA) fitted with a 34-G nanopass needle (Terumo, Tokyo, Japan). Then, 5 µL of 0.01% levofloxacin ophthalmic solution (Santen Pharmaceuticals Co., Ltd., Osaka, Japan) was applied topically.

Yasuda H., Tanaka M., Nishinaka A., Nakamura S., Shimazawa M, & Hara H. (2021). Role of Activating Transcription Factor 4 in Murine Choroidal Neovascularization Model. International Journal of Molecular Sciences, 22(16), 8890.

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Chemical Inhibitor Screening Protocol

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Tunicamycin (TM) and thapsigargin (TG) were obtained from Fujifilm-Wako (Osaka, Japan). GSK2656157 and ISRIB were obtained from Cayman Chemical (Ann Arbor, MI, USA). All other chemicals were obtained from Sigma.

Nishikawa S., Itoh Y., Tokugawa M., Inoue Y., Nakashima K.I., Hori Y., Miyajima C., Yoshida K., Morishita D., Ohoka N., Inoue M., Mizukami H., Makino T, & Hayashi H. (2019). Kurarinone from Sophora Flavescens Roots Triggers ATF4 Activation and Cytostatic Effects Through PERK Phosphorylation. Molecules, 24(17), 3110.

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Macrophage Polarization and Tumor Co-culture

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Bone marrow cells were differentiated in the presence of recombinant mouse M-CSF (20 ng/ml; PeproTech) in complete medium (RPMI 1640 containing 10 mM glucose, 2 mM L-glutamine, 100 U/mL of penicillin/streptomycin and 10% FBS) for 7 days. Fresh complete medium with M-CSF was supplemented on day 6 prior to use. Day 7 macrophages were washed and variously stimulated with IL-4 (20 ng/ml; PeproTech) or LPS (20 ng/ml; Sigma-Aldrich) plus IFNγ (50 ng/ml; PeproTech) in the absence or presence of 5 μM GSK2656157 (Cayman Chemical), 30 μM CBR-5884 (Cayman Chemical), or 5 μM ISRIB (Cayman Chemical). Macrophages were harvested after 24 h and analyzed by flow cytometry for the expression of M2 or M1 activation. For tumor co-culture experiments, macrophages and tumor cells were collected on day 7. Tumor cells were plated at a density of 5-6 x 10⁵ in a 12-well plate for at least 1 h prior to addition of macrophages. Once tumor cells were attached, 2 x 10⁵ macrophages were added to each well for 72 h. For some experiments, macrophages were cultured with IL-4 in the presence of absence of 1 mM dimethyl-α-KG (dm-KG; Sigma-Aldrich) or 25 μM GSK-J4 (Selleck Chem) for 6 h, and cells were harvested for the further experiments as indicated.

Raines L.N., Zhao H., Wang Y., Chen H.Y., Gallart-Ayala H., Hsueh P.C., Cao W., Koh Y., Alamonte-Loya A., Liu P.S., Ivanisevic J., Lio C.W., Ho P.C, & Huang S.C. (2022). PERK is a critical metabolic hub for immunosuppressive function in macrophages. Nature immunology, 23(3), 431-445.

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Laser-Induced Retinal Injury Treatment

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Timolol maleate 0.5%, latanoprost 0.005%, and vehicle solutions were kind gifts from Nitto Medic Co. Ltd. (Toyama, Japan), all of which were administered by eye drop (5 µl) immediately and at 3, 6, 12, and 18 h after laser irradiation. In another independent experiment, an integrated stress response inhibitor (ISRIB; 9 ng/2 µl; Cayman Chemical, Ann Arbor, MI) was injected (2 μl) into the vitreous body of the right eye immediately after laser irradiation using a sterile 34-gauge needle (Terumo, Tokyo, Japan) attached to a Hamilton glass syringe (701 N; Hamilton Co., Reno, NV). For controls, mice were IV injected with 2 μl of 0.01 M PBS into the right eye, after which 0.5% levofloxacin ophthalmic solution (Santen Pharmaceuticals Co., Ltd.) was applied topically to the treated eyes.

Fuma S., Hidaka Y., Nishinaka A., Yasuda H., Aoshima K., Nakamura S., Hara H, & Shimazawa M. (2023). Timolol maleate, a β blocker eye drop, improved edema in a retinal vein occlusion model. Molecular Vision, 29, 188-196.

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Investigating ISR Modulation in HeLa Cells

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HeLa cells were plated onto a 6-well plate in DMEM culture media supplemented with 10% FBS and penicillin-streptomycin and ISRIB (200 nM) (R and D Systems, cat # 5284/10). Minocycline (100 μM), thapsigargin (200 nM) (Cayman Chemical, cat # 10522) or different combinations of the three were added to 70% confluent cells and incubated for 3 hr (ISRIB and/or thapsigargin-treated samples) or 6 hr (Minocycline-treated samples). ³⁵S incorporation was measured using the protocol described above (³⁵S incorporation).

Solis G.M., Kardakaris R., Valentine E.R., Bar-Peled L., Chen A.L., Blewett M.M., McCormick M.A., Williamson J.R., Kennedy B., Cravatt B.F, & Petrascheck M. (2018). Translation attenuation by minocycline enhances longevity and proteostasis in old post-stress-responsive organisms. eLife, 7, e40314.

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