The overall complex size of IgA aggregates was determined either by SEC or SDS-PAGE. For evaluating the involvement of intermolecular disulfide connectivity in IgA complexes, IgA samples were treated with either reducing agents, such as DTT, TCEP, or reduced glutathione (MilliporeSigma), or interventional drugs, such as cysteamine (MilliporeSigma) and WR-1065 (MilliporeSigma). Before running on SEC, samples were treated with the drugs at indicated concentrations at 37°C for 1 hour.
Reduced glutathione
Manufactured by Merck Group
Sourced in United States, Germany, Czechia, Argentina, Sao Tome and Principe, China, India
Reduced glutathione is a biochemical compound that serves as an antioxidant in biological systems. It plays a key role in maintaining the redox state of cells and protecting them from oxidative stress.
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Lab products found in correlation
Trichloroacetic acid, by Merck Group (38 mentions)
Thiobarbituric acid, by Merck Group (37 mentions)
Oxidized glutathione, by Merck Group (36 mentions)
Bovine serum albumin (bsa), by Merck Group (32 mentions)
1 chloro 2 4 dinitrobenzene, by Merck Group (24 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (24 mentions)
Hydrogen peroxide, by Merck Group (23 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (20 mentions)
Glutathione reductase, by Merck Group (20 mentions)
Sodium hydroxide (naoh), by Merck Group (16 mentions)
Nitroblue tetrazolium, by Merck Group (14 mentions)
Ascorbic acid, by Merck Group (14 mentions)
Hydrochloric acid (hcl), by Merck Group (14 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (13 mentions)
Dithiothreitol (dtt), by Merck Group (13 mentions)
Nadph, by Merck Group (12 mentions)
Epinephrine, by Merck Group (10 mentions)
2 thiobarbituric acid, by Merck Group (10 mentions)
5 5 dithiobis 2 nitrobenzoic acid dtnb, by Merck Group (9 mentions)
Hydrogen peroxide h2o2, by Merck Group (9 mentions)
308 protocols using reduced glutathione
1
Evaluating IgA Aggregate Disulfide Bonds
2
Expression and Purification of PBRM1 Bromodomains
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pGST parallel expression plasmids with PBRM1 BDs were individually expressed in E. coli strain BL21. The expression of the protein was induced by 0.1 mM isopropyl-β-d-thiogalactoside (IPTG, I6758, Millipore Sigma) for 20 h at 18 °C. The GST-tagged proteins were purified with glutathione-Sepharose 4B (GE Healthcare, Catalog #17–5130–01) according to the manufacturer’s protocol and eluted with 10 mM reduced glutathione (G4251, Millipore Sigma). To remove glutathione and concentrate proteins, the eluates were spun using Amicon Ultra-15 Centrifugal Filter Unit (UFC901096, Millipore Sigma) with dialysis buffer (25 mM Tris-HCl (pH 8.0), 100 mM NaCl).
3
GST Pull-down Assay for UBASH3A PGM Domain
4
Preparation of Defined Starvation Media
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To prepare defined starvation medias, we reconstituted media containing the components of RPMI-1640, including the following reagents: 1XDPBS (Gibco, 14040–133), Phenol Red (Sigma-Aldrich, P3532), HEPES Buffer 1 M (Gibco, 15630–080), RPMI-1640 Vitamin Mix 100X (Sigma-Aldrich, R7256), Sodium bicarbonate (Sigma-Aldrich, S5761), reduced Glutathione (Sigma-Aldrich, G6013), 200 mM Glutamine (Gibco, 25030–081), D-Glucose (Fisher Scientific, D16), and dialyzed Fetal Bovine Serum (Sigma-Aldrich, F0392). Amino acid starvation was prepared by adding all components except amino acid mix and glucose starvation media was prepared by adding all components except glucose. See Supplemental Table 1 for full formulations. Medias were prepared, pH adjusted to 7.2–7.4, sterile-filtered, and stored at 4°C.
5
Recombinant GST-PGP3 Fusion Protein Purification
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Coding sequence 5 (CDS5) was amplified by PCR from recombinant plasmid pSP73-SW2 Recombinant GST-PGP3 (54 kDa) fusion proteins expressed using the pGEX-4T-1 vector were washed once more with PBS and bound GST-tagged protein was eluted using buffer A (50 mM reduced glutathione (Sigma-Aldrich), 50 mM Tris-HCl [pH 8]). Empty vectors of pGEX-4T-1 and pGEX-6P-1 were also induced using the same protocol to produce the GST fusion tag (26 kDa). Recombinant GST-PGP3 fusion proteins expressed using the pGEX-
6
Histidine Metabolism Pathway Analysis
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Water (18.2 MΩ cm À1 ) was purified with a Milli-Q Plus ® system (Millipore, Madrid, Spain). The LC-MS solvent (acetonitrile, ACN) and additive (formic acid) were of UHPLC-MS-grade and were from Scharlau (Scharlab S.L., Barcelona, Spain). L-glutamate, imidazole-4-acetate, N-acetylhistamine, cis-urocanate, imidazole propionate and 2-amino-4-thiazoleacetic acid were from Sigma-Aldrich Chemie GmbH. Trans-urocanate, histamine, and atenolol were obtained from Acros Organics (Geel, Belgium).
F I G U R E 1 Metabolic pathways involving measured metabolites from the histidine metabolism L-histidine was from VWR International Ltd (UK). Other chemicals used for measuring enzymatic activities were BugBuster Protein Extraction Reagent (Novagen, Darmstadt, Germany), magnesium chloride, reduced glutathione, pyridoxal-5 0 -phosphate and aminoguanidine (Sigma-Aldrich, Chemie GmbH).
F I G U R E 1 Metabolic pathways involving measured metabolites from the histidine metabolism L-histidine was from VWR International Ltd (UK). Other chemicals used for measuring enzymatic activities were BugBuster Protein Extraction Reagent (Novagen, Darmstadt, Germany), magnesium chloride, reduced glutathione, pyridoxal-5 0 -phosphate and aminoguanidine (Sigma-Aldrich, Chemie GmbH).
7
Purification and Characterization of GST-Fusion Proteins
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HeLa cells were cultured according to American Type Culture Collection guidelines. Unless otherwise noted, all chemicals and reagents were purchased from Fisher Scientific (Pittsburgh, PA). Ribonuclearse-free deoxyribonuclearse and restriction endonuclearses were purchased from Promega (Madison, WI). Taq DNA polymerase and Vent DNA polymerase were purchased from New England Biolabs (Beverly, MA). Pfu Turbo DNA polymerase was purchased from Stratagene (La Jolla, CA). Superscript II reverse transcriptase (RT) and oligonucleotide primers were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA). 4′-6-Diamidino-wheel for 12 h. The beads with GST-IGFBP-5 and GST-importin-α5 were extensively washed with and resuspended in sonication buffer, and used for the GST pull-down assay. For the fusion protein purification, the beads with fusion proteins were freed from nucleic acid contamination using sonication buffer with 2 M NaCl, and the purified fusion protein was eluted with 100 mM Tris containing 20 mM reduced glutathione (Sigma, St. Louis, MO) [17] .
The pure protein was ultrafilterated and the protein purity and integrity were routinely checked by SDS-PAGE and silver staining/Coomassie blue, the amount of purified protein was determined using BCA protein assay kit (Pierce Biotechnology, Rockford, IL), and stored at -70 °C until use.
The pure protein was ultrafilterated and the protein purity and integrity were routinely checked by SDS-PAGE and silver staining/Coomassie blue, the amount of purified protein was determined using BCA protein assay kit (Pierce Biotechnology, Rockford, IL), and stored at -70 °C until use.
8
GST-tagged TARDBP Purification from E. coli
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GST-tagged recombinant TARDBP was expressed in BL21 (DE3) ECOSTM-competent E. coli cells (Nippon Gene, Tokyo, Japan) from the pGEX-TARDBP plasmid. Induction of protein expression was carried out with 0.5 mm isopropyl β-d-thiogalactopyranoside (IPTG) and cells were grown at 37 °C for 3 hours. Cell pellets were resuspended in a GST binding buffer consisting of 150 mM NaCl, 25 mM Tris (pH 8.0), 0.05% (v/v) NP40, 1 mM EDTA (+protease inhibitors) and 0.25 mg/ml of lysozyme. Lysates were centrifuged for 20 min at 20,000 × g, 4 °C following sonication on ice. The GST protein was precipitated using Glutathione Sepharose 4B according to the manufacturer’s instructions (GE Healthcare Life Sciences). Elution of protein from the beads was achieved using a GST elution buffer containing 10 mM reduced glutathione (Sigma), pH 8.0 dissolved in 50 mM Tris-HCl.
9
Recombinant Protein Purification Protocol
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REP1∆AD and the full-length CGA1 were synthesized by GENEWIZ (Nanjing, China). Recombinant Rep1∆AD-His, as well as its variants, and GST-Cga1 was expressed in E. coli BL21.Cells were induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 16 °C overnight. Pellets were resuspended in 25 mM Tris-HCl pH 8.0, 500 mM NaCl and lysed by sonication. For purification of His-tagged proteins, lysates were applied to a Ni-NTA column (Qiagen),washed with 20 column bed volumes (CV) of resuspension buffer with 50 mM imidazole, and eluted with 5 CV of elution buffer (25 mM Tris-HCl pH 8.0, 300 mM NaCl and 250 mM imidazole). For GST pull down experiments, lysates were applied to Glutathione-Agarose (Sigma),washed with 20 CV of resuspension buffer with 1% Tween-20, and eluted with 5 CV of elution buffer (10 mM reduced glutathione [Sigma], 50 mM Tris-HCl, pH 9.0). Protein purity was determined via SDS–PAGE (Fig. S4D & S5C).
10
Kinetic Assay for Tryptophan Inhibitors
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The assay was carried out in a cuvette (1 cm optical path length) in a BioSpectrometer Kinetic (Eppendorf, Hauppauge, NY) at 37 °C as previously reported70 (link), but with some modification. The reaction mixture contained 0.005% BSA (Sigma), 1 mM reduced glutathione (Sigma), 100 μM pyridoxal phosphate (Sigma), 100 μM NADH (Sigma), 3 U/mL lactate dehydrogenase (Sigma) and apotryptophanase (10 U/mL, Sigma) in 0.15 M potassium phosphate buffer (pH 8.0). Following a 3 min incubation with inhibitor, the enzymatic reaction was initiated by adding l -tryptophan (300 μM final, Sigma). The decrease in optical density at 340 nm was monitored for 5 min at 5 s intervals and used for calculating the TIL reaction velocity. The inhibitory effect of TPL inhibitors (2-aza-tyrosine and l -meta-tyrosine) on TIL enzymatic activity was measured. Homo-BZI-Ala was used as a positive control of TIL inhibitor.
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