JEG-3 cells were infected with ZIKV for 1 h in DMEM (Gibco) with 2% FCS at a multiplicity of infection (MOI) of 2.0. Virus was aspirated and fresh culture medium added. Infected cells were cultured for the indicated times before harvesting for assay. In some cases, JEG-3 cells were pretreated with 10 μM salubrinal (Sigma) for 1 h before infection, and salubrinal was retained in the culture medium post infection. JEG-3 cells were infected with MOI 0.5 HSV-2 for 1 h, then medium was aspirated and cells cultured in fresh medium for 1 or 2 days. For HCMV infection, cells were spinoculated (1 h, 2,800 rpm) with MOI 4.0 virus, which was maintained during 12 h of culture at 37 °C. Spinoculation and culture were repeated once or twice to enhance infection rates, and then medium was aspirated and cells cultured in fresh medium for 12 h before harvesting. In some experiments, cells were treated with 0.5 μg ml–1 tunicamycin for 24 h, with or without salubrinal pretreatment 1 h earlier. salubrinal was retained in culture throughout tunicamycin treatment.
Salubrinal
Manufactured by Merck Group
Sourced in United States, Germany, Sao Tome and Principe
Salubrinal is a laboratory compound that inhibits the dephosphorylation of eIF2α, a key regulator of protein synthesis and cellular stress response. It is commonly used in research to investigate the effects of endoplasmic reticulum stress and the unfolded protein response in cellular systems.
Automatically generated - may contain errors
Lab products found in correlation
Tunicamycin, by Merck Group (7 mentions)
Thapsigargin, by Merck Group (7 mentions)
Gsk2606414, by Merck Group (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Annexin 5 fitc pi apoptosis detection kit, by BD (3 mentions)
Rapamycin, by Merck Group (3 mentions)
Cycloheximide (chx), by Merck Group (3 mentions)
Z devd fmk, by Merck Group (2 mentions)
Z vad fmk, by Merck Group (2 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (2 mentions)
β actin antibody, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Biowest (2 mentions)
Chloroquine, by Merck Group (2 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (2 mentions)
Cisplatin, by Merck Group (2 mentions)
Eif2α, by Cell Signaling Technology (2 mentions)
Cycloheximide (chx), by Avantor (2 mentions)
Fetal bovine serum (fbs), by Omega Scientific (2 mentions)
Isrib, by Merck Group (2 mentions)
52 protocols using salubrinal
1
Viral Infection Assays in JEG-3 Cells
2
Apoptosis Mechanism Investigation Protocol
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Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Biowest (Nuaillé, France). Polyvinylidene difluoride (PVDF) membranes and goat anti-rabbit and horseradish peroxidase-conjugated immunoglobulin (Ig) G were obtained from Millipore (Bellerica, MA, USA). MTT, 3-MA, salubrinal, Z-DEVD-FMK, Z-VAD-FMK and β-actin antibody were purchased from Sigma (St. Louis, MO, USA). An annexin V-FITC/PI apoptosis detection kit was purchased from Pharmingen (San Diego, CA, USA). Enhanced chemiluminescence (ECL) reagents were purchased from Pierce Biotechnology (Rockford, IL, USA).
3
Characterization of Zip7 Inhibitors
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NVS-ZP7-4 is an inhibitor of Zip7 that was derived from a compound (NVS-ZP7-1) that was obtained in a phenotypic screen of inhibitors of Notch signaling as previously described [23 (link)]. NVS-ZP7-6 is an inactive analog of NVS-ZP7-4. NVS-ZP7-1, NVS-ZP7-4 and NVS-ZP7-6 were synthesized internally at Novartis. Tauroursodeoxycholic acid (TUDCA) (cat# T0266), salubrinal (cat# 324895), dibenzazepine (cat# 209984-56-S) and compound E (cat# 209986-17-4) were purchased from Sigma. All recombinant proteins were purchased from R&D Systems, Minneapolis, MN, unless otherwise noticed. TPEN (N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine) was purchased from Sigma (cat# P4413). DTPA (diethylenetriaminepentaacetic acid) was purchased from Sigma (cat# D1133). The following antibodies were purchased from Cell Signaling Technologies and used at 1:1000 dilution: BiP (C50B12) rabbit mAb #3177, IRE1 α (14C10) rabbit mAb #3294, CHOP (L63F7) mouse mAb #2985, calreticulin (D3E6) rabbit mAb #12238, PERK (C33E10) rabbit mAb #3192, XBP-1s (D2C1F) rabbit mAb #12782, ZIP7/SLC39A& (D103A) rabbit mAb # 33176. The following antibodies were purchased from Cell Technologies and used at 1:2000 dilution: GAPDH (14C10) rabbit mAb #2118, β -actin (8H10D10) mouse mAb #3700, goat anti-rabbit HRP-linked IgG #7074, horse anti-mouse HRP-linked IgG #7076.
4
Cell Signaling Pathway Modulation
5
Transient transfection of tau and HSF1 constructs
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Plasmid constructs used in transient transfection include full length tau (pcDNA-WT, R406W) and repeated domain tau constructs (pcDNA-TauRD WT, TauRD P301L, TauRD ΔK280); pcDNA-HSF1 WT, S303A and HSF1 Δ156–226; pcDNA-HSP70a5 (BiP/GRP78). HSF1 WT and HSF1 S303A were generously given by Dr. Dennis Thiele at Duke University. HSF1 Δ156–226 (trimerization mutant) was cloned by using a method of site-directed mutagenesis (Agilent Technologies). Tunicamycin, thapsigargin, salubrinal, resveratrol, piceid, celastrol, rapamycin, and riluzole were all purchased from Sigma. siRNA oligomers for CHOP and BiP were purchased from Sigma.
6
Oligonucleotide and Small Molecule Protocols
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All oligonucleotide sequences and modification patterns synthesized in-house are summarized in Supplementary Table S1. Accell siRNA (Thermo Fisher) targeting HTT was purchased and used without further purification. Clonidine (Sigma–Aldrich), Retro-1 (Sigma–Aldrich) and Salubrinal (Sigma–Aldrich) were dissolved in DMSO and stored as 10 mM stock solutions at −20°C. Unless otherwise specified, all oligonucleotides and small molecules were diluted in OptiMEM (Gibco) prior to administration in vitro.
7
Inhibition of Cell Proliferation and Apoptosis Induction
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JNJ-10198409 (JNJ) and Picropodophylin (PPP) were purchased from Calbiochem, Merck Millipore (Nottingham, UK). Propidium iodide, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), PDGF-BB, IGF-1, nocodazole, chloroquine, AEBSF, Ucf-101 and salubrinal were purchased from Sigma-Aldrich (St. Louis, MO, USA), 7-Amino-Actinomycin D (7-AAD) was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Z-VAD (OMe)-FMK (ICn), Z-D(OMe)E(Ome)VD(OMe)-FMK (IC3), Ac-IETD-CHO (IC8) and necrostatin-1 were purchased from Calbiochem, Merck Millipore (Darmstadt, Germany). MK-1775 was purchased from Selleckchem (Houston, TX, USA).
8
Establishment of Radioresistant OSCC Cell Lines
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The human HPV‐negative OSCC cell lines FaDu and Detroit562 were cultured in minimum essential medium containing 10% heat‐inactivated fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL at 37°C and 5% CO2.
The radioresistant FaDu and Detroit562 cell lines were established according to a previously published protocol.12 Briefly, 1000 cells/cm2 FaDu and Detroit562 cells were seeded in six‐well plates and allowed to attach for 24 hours before receiving 4 and 3 Gy of irradiation, respectively. The cells were then cultured for 10‐14 days to form colonies. The culturing and irradiation process was repeated four times with single‐cell clones. The resultant radioresistant cells are denoted as FaDuR and Detroit562R, whereas non‐irradiated parental cells are denoted as FaDuP and Detroit562P.
Ly294002, 3‐Methyladenine (3‐MA), and salubrinal were all purchased from Sigma (St Louis, MO, USA). Tunicamycin was purchased from Abcam (Cambridge, UK). Erbitux (cetuximab) was purchased from Merck (Darmstadt, Germany).
The radioresistant FaDu and Detroit562 cell lines were established according to a previously published protocol.
Ly294002, 3‐Methyladenine (3‐MA), and salubrinal were all purchased from Sigma (St Louis, MO, USA). Tunicamycin was purchased from Abcam (Cambridge, UK). Erbitux (cetuximab) was purchased from Merck (Darmstadt, Germany).
9
Synthesis and Characterization of NEO214
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Rolipram, bortezomib, and forskolin were obtained from LC Laboratories (Woburn, MA, USA). Perillyl alcohol, salubrinal, piclamilast, cycloheximide, and DMSO were from Sigma-Aldrich (St. Louis, MO, USA). NEO214 was manufactured by Norac Pharma (Azusa, CA, USA) and was provided by NeOnc Technologies, Inc. (Los Angeles, CA, USA). Chemical purity was 99%, which was confirmed by 1H-NMR (300 MHz), mass spectrometry, and microanalysis. The white solid was dissolved in DMSO at 100 mM, and aliquots were stored at –80 °C. NEO214’s IUPAC name is ((S)-4-(prop-1-en-2-yl)cyclohex-1-enyl)methyl 4-(3-(cyclopentyloxy)-4-methoxyphenyl)-2-oxopyrrolidine-1-carboxylate and its CAS number is 1361198-80-2. Molecular formula is C27H35NO5 and molecular weight is 453.57 g/mol.
10
Modulation of Cellular Stress Responses
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Thapsigargin was obtained from Enzo Life Sciences. The TLR4 agonist KLA (Kdo2-Lipid A) was from Santa Cruz Biotechnology. Sodium arsenite, the oxindole-imidazole-C16, salubrinal, the three flavonoids luteolin, myricetin and quercetin were from Sigma. The PERK inhibitor GSK2606414 (Perki) was from Calbiochem. The PKR and PERK inhibitors, the flavonoids and salubrinal were all kept in DMSO as stock solution then diluted in cell culture medium immediately before use. The CHAPS detergent (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate was from Sigma. Control siRNA (ON-TARGETplus Non-targeting Pool #D-001810-10-0X) and siRNA against PKR (ON-TARGETplus EIF2AK2 siRNA LQ-003527-00-0002) were from Dharmacon Research, Inc. (Lafayette, CO). siRNA against PACT (PRKRA, ID:s16335, Ambion; Life Technologies) was a kind gift of A. Komarova (Institut Pasteur).
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