Golgistop containing monensin

Bone marrow-derived macrophages were primed with 1 ng/ml LPS for 3 days and stimulated with 100 ng/ml LPS for 7 h. Monensin-containing GolgiStop (BD Biosciences) was added for the last 6 h of culture. After washing, cells were lysed in lysis buffer (4% SDS, 50 mM TEAB pH 8.5, 10 mM TCEP), boiled and sonicated with a BioRuptor (30 cycles: 30 s on, 30 s off) before alkylation with 20 mM iodoacetamide for 1 h at room temperature in the dark. Lysates were subjected to the SP3 protein clean-up procedure (22 (link)), eluted into digestion buffer (0.1% SDS, 50 mM TEAB pH 8.5, 1 mM CaCl2) and digested with trypsin at a 1:50 (enzyme:protein) ratio. TMT labeling and peptide clean-up were performed according to the SP3 protocol. Samples were eluted into 2% DMSO, combined, and dried under vacuum. TMT samples were fractionated using offline high pH reverse-phase chromatography. Peptides were separated, concatenated to 22 fractions, dried and peptides redissolved in 5% formic acid and analyzed by LC-MS.

For intracellular cytokine analysis, single cell suspensions (0.5-1 million cells per well) were stimulated in 24 well plates with complete RPMI containing 20 nM phorbol myristate acetate (PMA; Sigma-Aldrich) and 1μg/ml ionomycin (Sigma-Aldrich), at 37°C for 4 hours. After 1 hour of incubation, 1μl/ml of GolgiStop (containing monensin; BD Biosciences) was added to each well. Surface stained cells were stained for intracellular antigen following Fixation/Permeabilisation (Foxp3-staining kit, eBiosciences). Intracellular antibodies consisted of anti-Ki67-FITC (B56; BD Bioscience) or anti-Ki67-BrilliantViolet 421 (16A8; BioLegend), anti-IL-2-PE (JES6-5H4; eBioscience), anti-Foxp3-PE-Cy7 (FJK-16s; eBioscience), and anti-T-bet-PerCPCy5.5 (eBio4B10; eBioscience). For flow cytometric analysis, samples were acquired on a FACS Canto II flow cytometer (BD Biosciences), and were subsequently analysed using FlowJo software.

PBMCs from healthy donors’ fresh blood were cultured in RPMI 1640 medium (Gibco) containing 10% of FBS and stimulated 50% (v/v) with supernatant from macrophages treated with 1 mM of SB and PBA. Stimulation was performed for 6 h at 37 °C 5% CO₂ in the presence of Golgi-Plug containing Brefeldin A (BD Biosciences, San Jose, CA, USA) and Golgi-Stop containing Monensin (BD Biosciences) added after 1 h of the stimulation according to the manufacturer’s instructions. After that, PBMCs were washed and stained with the Live/Dead fixable Blue (Invitrogen) and surface markers (listed in Supplementary Table S1) for 30 min at room temperature, twice washed, fixed and permeabilized using the eBioscience™ Transcription Factor Fixation/Permeabilization (Invitrogen) according to the manufacturer’s instructions. Subsequently, the fixed and permeabilized PBMCs were staining using fluorochrome-conjugated antibodies against intracellular makers listed in Supplementary Table S1. Labeled cells were acquired on a Cytek Aurora Spectral Cytometer (Cytek Biosciences, Fremont, CA, USA). Data were analyzed using FlowJo (TreeStar, Ashland, OR, USA) v10.6.2 software.

Peripheral blood mononuclear cells (2 × 10⁵ cells in 200 μl) were cultured in 96-well U-bottom plates in complete medium with or without low concentration of cytokines [LCC; 12·5 pg/ml recombinant human (rh) IL-12 (PeproTech, Rocky Hill, NJ) plus 10 ng/ml rhIL-18 (MBL, Woburn, MA)]; high concentration of cytokines (HCC; 5 ng/ml rhIL-12 plus 50 ng/ml rhIL-18); or 7·5 μg/ml tetanus toxoid (TT), 1 μg/ml diphtheria toxoid (DT) or 1 IU/ml whole cell pertussis (all from the National Institute for Biological Standards and Control, London, UK) for 18 hr at 37°. GolgiPlug (containing Brefeldin A, 1/1000 final concentration; BD Biosciences, Oxford, UK) and GolgiStop (containing Monensin, 1/1500 concentration; BD Biosciences) were added after 15 hr.

The heterologous HA-His- or MBP-HA-His-tagged protein CLEC-47 from C. elegans was expressed in HEK 293 cells, and secretion was confirmed by immunoblotting. Cells were seeded into a 6-well plate 1 day before transfection. pcDNA4-based plasmids were transfected with Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). The medium was replaced with Opti-MEM I reduced-serum medium (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 6 h posttransfection. Cells were collected and lysed with radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Thermo Fisher Scientific).
Recombinant proteins in total cell lysates and conditioned culture medium were separated on an 8% to 16% Mini-Protean TGX stain-free protein gel (Bio-Rad, Hercules, CA, USA) and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad), which was blocked with a membrane-blocking solution (Invitrogen). HA tag monoclonal antibody (12CA5) (Invitrogen) was utilized for detection. The membrane was developed with an enhanced chemiluminescence (ECL) system, and images were acquired with ChemiDoc imaging systems (Bio-Rad).
The inhibition of secretion was performed by adding manufacturer-recommended amounts of brefeldin A (BFA) or GolgiStop (containing monensin) (BD Biosciences, San Jose, CA, USA) to the changed Opti-MEM I reduced-serum medium.