Dithiotreitol

Frozen aliquots of bacterial pellets were thawed and introduced into 1.5 mL Eppendorf tubes, containing a mixture of glass beads and 150 μL of 50 mM NH₄HCO₃, 5 mM dithiotreitol (Sigma-Aldrich-Fluka), pH = 8.0. The tubes were placed on an ultrasound probe (Hielscher Ultrasonics GmBH, Teltow, Germany) and the samples were disrupted during 5 min. They were further alkylated with 12.5 mM iodoacetamide (Sigma-Aldrich-Fluka) during 5 min in the dark. Trypsin (10 μg) (Sigma-Aldrich-Fluka) was added to the samples and the digestion was performed on a heating block at 50°C during 15 min. The tryptic digestion was stopped by acidifying samples with 0.5 μL formic acid (Sigma-Aldrich-Fluka). The samples were desalted using Oasis HLB 3 cm³ (60 mg) reversed phase cartridges (Waters, Milford, MA). The cartridges were conditioned with 1 mL of methanol (Merck Millipore, Billerica, MA), then with 1 mL of water (LC-MS grade, Fisher Scientific, Strasbourg, France) containing 0.1% formic acid prior to loading of the tryptic digest. Cartridges were washed with 1 mL of water containing 0.1% formic acid and eluted with methanol/water (80:20, v/v) containing 0.1% formic acid. The samples were dried by vacuum centrifugation, suspended in 200 μL of water/acetonitrile (95/5, v/v) containing 0.5% of formic acid and directly analyzed or stored at −20°C until analysis.

Normal human kidney epithelial cells of proximal origin (HK-2) were purchased from ATCC/LGC Standards (lot number 60352186), and cultured according to previously published method.⁴⁷ (link) HK-2 is a cell line derived from primary proximal tubule cells. HK-2 cells are cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 5 μg/mL insulin, 10 μg/mL human apotransferrin, 500 ng/mL hydrocortisone, 10 ng/mL epithelial growth factor, 6.5 ng/mL triiodothyronin, 5 ng/mL sodium selenite, 1% fetal calf serum, 25 IU/mL penicillin, 25 μg/mL streptomycin and 10 mM HEPES buffer. These cells lines are Mycoplasm free (Mycoalert Mycoplasma Detection Kit, Lonza).
Tunicamycin, thapsigargin, dithiotreitol, brefeldin A, actinomycin D, cyclosporine, DMSO and IL-6 were purchased from Sigma Aldrich. OSM was purchased from R&D systems. KIRA6 was purchased from Millipore. RSL3 and Stattic were purchased from MedChem Express.

N-(1-pyrene) maleimide, Alexa488 SE were obtained from Molecular Probes (Eugene, OR). DNase1, ATP, ADP, dithiotreitol (DTT), histone type III (histone mixture) and deoxyribonucleic acid (DNA) from calf thymus were purchased from Sigma Chemical Co. (St Louis, MO). Acetone dry powder was purchased from Pel-freeze Biologicals (Rogers, AR). Human recombinant H2A histone was bought from New England Bio Labs (Ipswich, MS). Viscous Aqua was purchased from Ursa BioScience (Abingdon, MD). Yeast cofilin was a generous gift of Prof. Emil Reisler, (University of California, Los Angeles, CA)

Lysozyme from chicken egg white (about 100,000 U/mg), α-chymotrypsinogen A from a bovine pancreas, ribonuclease A from a bovine pancreas (Type XII-A, 75–125 Kunitz units/mg protein), l-cysteine, cysteamine, l-glutathione, oxidized glutathione, N-acetyl-l-cysteine, cysteinylglycine, l-cysteine ethyl ester, 1-chloro-2,4-dinitrobenzene, 5,5′-dithiobis(2-nitrobenzoic acid), 4-chloro-7-nitrobenzofurazane, dithiotreitol, ethylendiamminotetreaacetic acid, urea, 8-anilinonaphthalene-1-sulfonic acid and all of the other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Acetonitrile and water (LC-MS grade) were obtained from Fisher Scientific (Strasbourg, France). Formic acid, dithiotreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate and porcine trypsin were purchased from Sigma-Aldrich-Fluka (Lyon, France). Blood culture bottles and agar plates for bacterial isolation and culture were obtained from bioMérieux (Marcy L’Etoile, France). Peptides were synthesised using Fmoc chemistry on a MultiPep RS peptide synthesiser from Intavis Bioanalytical Instruments AG (Koeln, Germany).

iIELs from 8-to-14 week old mice were isolated as follows. Briefly, intestines were removed and cleaned of mesenteric fat. Peyer's patches were excised, and intestines were rinsed with DPBS to clear intestines of fecal content. Subsequently, intestines were opened longitudinally, cut into 0.5-cm pieces, and transferred to a 50-ml conical tube. Intestines were incubated twice for 20 minutes at 37°C in Ca/Mg-free Hank's Balanced Sodium Solution (HBSS; Invitrogen, Carlstad, CA, USA) containing 5% FCS, 1 mM ethylenediaminetetraacetic acid (EDTA; Invitrogen) and 1 mM dithiotreitol (Sigma-Aldrich) at slow rotation. Cell suspensions were passed through a 40-µm cell strainer (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) and pelleted by centrifugation at 480 g. Pellets were resuspended in 44% Percoll (GE Healthcare, Buckinghamshire, UK) on an underlay of 67% Percoll, and centrifugated for 20°C at 2000 g. iIELs were collected from the 44%/67% Percoll interface, washed twice with phosphate buffered saline (PBS) for 5 minutes at 840 g, and resuspended in RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 100 µM non-essential amino acids (all Invitrogen) and 50 µM 2-mercaptoethanol (Sigma-Aldrich).

Acetonitrile (LC-MS grade), formic acid (LC-MS grade), iodoacetamid (purity ≥ 99%), dithiotreitol (purity ≥ 99%), sex hormone-binding globulin from human serum, bovine fetuin, and SOLu-Trypsin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hemopexin and haptoglobin standards from human plasma were purchased from Athens Research and Technology, Inc. (Athens, GA, USA). Water (LC-MS grade), ammonium hydrogen carbonate (LC-MS grade), and acetic acid (LC-MS grade) were supplied by Merck (Darmstadt, Germany). α2–3,6,8,9 neuraminidase, and GlycoBuffer 1 were purchased from New England BioLabs (Ipswich, MA, USA).