HEK293T were obtained from ATCC (Manassas, VA, USA). KBM-7 and HAP1 were obtained from T. Brummelkamp. HeLa were provided by M. Hentze, Jurkat E6.1 by W. Ellmeier. KBM7 TNFR1- cells were obtained from Haplogen (P00371D06). Cells were cultured in DMEM (Gibco, Grand Island, NY, USA), RPMI (Gibco) or IMDM medium (Gibco) supplemented with 10% (v/v) FBS (Gibco) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) (Gibco). Cell lines were checked for mycoplasma by PCR or ELISA. The reagents used were as follows: recombinant human TNF-α (300-01A, Peprotech), SMAC mimetic birinapant (S7015, Selleck Chemicals), FasLigand (ALX-522-020-C005, Enzo), recombinant human TRAIL (310-04, Peprotech), z-VAD-FMK (AG-CP3-0002, Adipogen), necrosulfonamide (480073, Merck Millipore), necrostatin-1 (N9037, Sigma-Aldrich), RIPK1 Inhibitor II 7-Cl-ONec-1 (Nec-1s) (504297, Merck Millipore), PNGase F (P0704, NEB), Endo H (P0702, NEB), MG-132 (S2619, Selleckchem), Thapsigargin (P9033, Sigma-Aldrich), Brefeldin A (1231, Tocris), Tunicamycin (T7765, Sigma Aldrich), and doxycycline (D9891, Sigma-Aldrich, St.Louis, MO, USA).
Brefeldin a
Manufactured by Bio-Techne
Sourced in United Kingdom
Brefeldin A is a lab equipment product offered by Bio-Techne. It is a fungal metabolite that inhibits the action of guanine nucleotide-exchange factors, which are responsible for the activation of ADP-ribosylation factors. This disrupts the function of the Golgi apparatus, leading to the accumulation of secretory proteins within the endoplasmic reticulum.
Automatically generated - may contain errors
Lab products found in correlation
Tunicamycin, by Merck Group (2 mentions)
Cycloheximide (chx), by Bio-Techne (2 mentions)
Necrosulfonamide, by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Nec 1, by Merck Group (1 mentions)
Imdm medium, by Thermo Fisher Scientific (1 mentions)
Birinapant, by Selleck Chemicals (1 mentions)
Recombinant human trail, by Thermo Fisher Scientific (1 mentions)
Recombinant human tnf α, by Thermo Fisher Scientific (1 mentions)
Fas ligand, by Enzo Life Sciences (1 mentions)
Z vad fmk, by Adipogen (1 mentions)
Endo h, by New England Biolabs (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Necrostatin 1, by Merck Group (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
10 protocols using brefeldin a
1
Cell Line Culture and Reagents
2
TRAIL and DR5-B Protein Expression and Purification
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Recombinant proteins TRAIL (amino acid residues 114–280) and its DR5-selective mutant variant DR5-B were expressed in Escherichia coli and purified in our laboratory as previously described (Yagolovich et al., 2019 (link)). Brefeldin A and cycloheximide were purchased from Tocris (Bristol, United Kingdom). Pan-caspase inhibitor Z-VAD-FMK was from Santa Cruz Biotechnology (Dallas, TX, United States).
3
Immunocytochemistry for Liver Cells
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The BMELs were treated 2 h prior to fixing with Brefeldin A (10ug/mL,Tocris 1231), an inhibitor of protein translocation to Golgi, to retain OPN inside the cells. The cells were fixed with 4% w/v PFA in 1XPBS solution at 72-h timepoint starting after the 6 h seeding time. This was followed by a 10-min permeabilization step using 0.25%TritionX/1X PBS solution and a 45-min blocking step with the blocking buffer (5% donkey serum in 0.25%TritonX/1X PBS solution). Both the primary and secondary antibody staining was either done overnight at 4 °C or for 1 h at room temperature in dark, with 3x 5 min 1X PBS washes in the middle. The antibody cocktail was prepared in the blocking buffer. Primary antibody anti-HNF4a (ab41898) was used at 1:200 dilution, anti-OPN (AF808) was used at 1:50 dilution and anti-E-Cad (AF748-SP) was used at 1:50. Actin was stained using fluorescent antibody Acti-Stain (PHDH1-A) at 1:700 dilution. All secondary antibodies (anti-mouse: ab98795, anti-goat: ab96935) were used at 1:50 dilution. For 12 mm coverslips, 30 µL of antibody cocktail was used to stain. Samples were then mounted in DAPI Flourmount G (Southern Biotech 0100-20).
4
Modulating SNAT2, GORASP2 and GADD34 in Cells
5
Pharmacological Modulation of CTL Recognition
6
BMP-7 Expression in Cultured Cells
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105 cells were seeded to Petri dishes and cultured for 6 days in CM or OM in the presence or absence of 100 ng/mL doxycycline. After 6 days, protein transport from the endoplasmic reticulum to the Golgi apparatus was inhibited by the addition of 10 µg/mL Brefeldin A (Tocris Bioscience, Tocris, United Kingdom). 24 h later, the cells were lysed with radioimmunoprecipitation assay (RIPA) buffer. The protein concentration was determined by Pierce BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA, USA), then 10 µg lysates from each sample were resolved by 14% SDS-PAGE analyzed by western blot using BMP-7 (6E5D12) antibody (Santa Cruz Biotechnology, Dallas, TX, USA) and Anti-GAPDH Antibody (Lifespan Biosciences, Seattle, WA, USA) respectively.
7
Pharmacological Treatments in Zebrafish Larvae
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All pharmacological treatments were performed for four hours unless otherwise specified. All drugs were washed out 3 times with E3. Metronidazole (MTZ) (Sigma, M3761) was made fresh daily as a 1M stock in DMSO stored at room temperature and is protected from light. MTZ was added to E3 medium at a concentration of 10mM. ISO-1 (Tocris, 4288), 4-IPP (Tocris, 3429), and Brefeldin A (Millipore-Sigma, B7651-5MG) were dissolved in DMSO and stored at 10mM stocks at −20°C. ISO-1 was added to E3 medium at a concentration of 60uM, 4-IPP and Brefeldin A are added at a concentration of 10uM. Dexamethasone (Tocris, 1126) was stored at −20°C as a 100mM stock, and replaced on a monthly basis. 4dpf larvae were treated at a concentration of 100uM. The Human Anti-CD74 antibody (BioLegend, 326802) was stored at 4°C, and added to E3 at a 1:400 dilution.
8
Neuronal Cell Culture and Receptor Assays
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Brefeldin A and mecamylamine hydrochloride was purchased from Tocris Bioscience (Bristol, United Kingdom). [125I]Epibatidine (2200 Ci/mmol) was purchased from Perkin-Elmer Life Science (Whaltam, MA). 2-(2-bromoacetyloxy)-N,N,N-trimethylethanaminium bromide (BrACh), cytisine, cytosine β-d -arabino-furanoside (ARA C), 5,5′-dithio-bis(2-nitrobenzoic acid (DTNB), dihydro-β-erythroidine, 1,4-dithio-dl -treithol (DTT), (-)-nicotine hydrogen tartrate, polyethyleneimine (PEI), and poly-l -lysine (>30,000 kDa) were purchased from Sigma Aldrich Chemical Company (St. Louis, MO). The 4-(2-Hydroxyethyl)-piperazineethanesulfonic acid (HEPES) half-sodium salt was from Roche Diagnostics Corporation (Indianapolis, IN). Neurobasal media, Minimal essential media (MEM), B27 supplement, Glutamax™, inactivated horse serum, and TrypLE express were purchased from Invitrogen (Carlsbad, CA).
9
Intracellular Cytokine Staining for T Cells
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Following T cell culture, cells were resuspended in media containing phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Fisher Scientific), ionomycin (750 ng/mL, Fisher Scientific) and brefeldin A (5 ug/mL, Tocris Bioscience) for 4 hours. Cells were then fixed, permeabilized and stained with fluorescent-labelled antibodies for CD4, FoxP3, IL-17 and IL-22 using FoxP3 Fix/Perm Buffer Set (Biolegend). Cells were analyzed on a Novocyte 3000 flow cytometer (Acea Biosciences, Inc.), with data analyzed using FlowJo software.
10
Comprehensive PBMC Immunophenotyping Protocol
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PBMCs were isolated using LSM (MP Biomedicals) from patients and healthy individuals. For intracellular staining, cells were plated with complete RPMI containing phorbol myristate acetate (PMA 50 ng/mL; Sigma-Aldrich), ionomycin (500 ng/mL; Sigma-Aldrich) and Brefeldin A (8 ng/mL; Tocris Bioscience) for 4 hours. Cells were fixed and permeabilised with the eBioscience Foxp3 staining kit (eBioscience). Anti-human antibodies included anti-NFIL3 (REA732) (Miltenyi Biotec), anti-CD14 (TuK4) (eBioscience); anti-CD3 (Miltenyi Biotec); anti-CD16 (3G8), anti-CD56 (NCAM16.2), anti-CD123 (7G3), anti-CD27 (L128), anti-CD45RA (HI100), anti-CD8 (SK1), anti-CD4 (SK3), anti-CD1c (L161), anti-IFNγ (4S.B3), anti-T-BET (4B10), anti-IL-17a (N49-653), anti-GATA3 (L50-823) (all from BD Biosciences); anti-HLA-DR (L243), anti-CD19 (HIB19), anti-CD56 (NCAM16.2), anti-CD11c (3.9), anti-CCR7 (G043H7), anti-FOXP3 (206D), anti-RORγt (Q21-559), anti-TNFα (MAb11), anti-IL-4 (MP4-25D2) (all from BioLegend); purified Rabbit-anti-human NFIL3 (D5K8O) (Cell Signaling Technology) followed by Donkey-anti-Rabbit-IgG (Thermo Fisher Scientific). Data were collected on BD Symphony (BD Biosciences) and analysed using FlowJo V.10.5 (Tree Star Inc.).
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