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. +4-°c-overnight, The plates were washed four times with washing buffer

. Subsequently, 200 ?l/well of blocking solution (1% BSA in PBS-Tween20) was added and plates were incubated for 2h at room temperature. After washing four times, 100 ?l/well of standard dilution of TRX80 (diluted in blocking solution) or samples were added in duplicates and incubated for 2h at room temperature. Thereafter, p.100

, ?l/well of rabbit polyclonal anti-TRX (N-term) (ABIN356853, antibodies-online GmbH) were added at

, ?g/ml and incubated for 2 h at room temperature. Following washing four times, 100 ?l/well of antirabbit IgG-peroxidase (A 6154, Sigma) were added and incubated for 1 h at room temperature

, Subsequently, plates were washed six times and 150 ?l/well of the substrate were added (1-Step? ABTS, 37615-ThermoScientific). After 30 min of incubation, 100 µl of stop solution (1% SDS in PBS) were added to each well and the absorbance was measured at 405 nm by a microplate reader, Colocalization of TRX80 and TNF-on M1 macrophages in human atherosclerotic lesions

, Human atherosclerotic vessel specimens from 3 patients undergoing vascular surgery for atherosclerotic complications were formalin-fixed, paraffin-embedded, and 5 µm sections were prepared for immunohistochemical analysis. M2 macrophages were visualized with anti-CD206 (Sigma, 1/500), TRX80 was stained with an antibody that recognizes only the truncated form of thioredoxin (IMCO Corp, Sweden, 1/500). M1 macrophages were stained with anti-TNF-antibody (Abcam, 1/250). In control samples, one of the first antibodies or both first antibodies were substituted with control IgG. For fluorescent immunostaining, Cy3-and Cy5-coupled secondary F(ab') 2 (Dianova) were used and visualized with Axioskop 2 plus fluorescence microscope

, Measurement of ADAM-10 and ADAM-17 activities

, glutamine (1%) (Invitrogen, France) and pooled human sera (10%) (Promo-Cell, Germany) at a density of 2×10 6 cells/well in 6-well Primaria-plastic culture dishes, Peripheral Blood Mononuclear Cells (PBMC) were isolated from buffy-coats of healthy young and aged donors

M. Cellytic? and . Reagent, The cell suspension was incubated on ice for at least 20 min and centrifuged for 10 min at 10,000 X g at 4°C. The supernatant was collected and used to assay ADAM-10 and ADAM-17 activities using respectively, Sigma) supplemented with a Protease Inhibitor Cocktail

, SensoLyte ® 520 ADAM10 Activity Assay Kit *Fluorimetric* (Anaspec, France) and InnoZyme? TACE Activity Kit

, After electroblotting onto PVDF Immobilon ® -P Transfer Membrane (Millipore, France), the blot was blocked with 5% non-fat dry milk in Tris Buffered Saline (TBS) for 1 h and incubated overnight at 4°C with specific antibodies, Western blotting PBMC were cultured, lysed and centrifuged as previously described. Proteins (20 ?g/lane), evaluated using Pierce? BCA Protein Assay Kit from Thermo Scientific, were separated on Criterion? TGX? Precast Gels 4-15%, p.5000

, For capillary tube formation, cells were seeded (30,000 cells/well) on 24-multiwell plates coated with Matrigel (BD Biosciences) and incubated in containing 10% (v/v) fetal bovine serum (FBS), 2 mmol L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin, IL-4 (10 ng/ml) or recombinant human TRX1 (rhTRX1, 1 µg/mL) or rhTRX80 (1 µg/mL) (all from R&D Systems) or a combination thereof

, Blood was collected into EDTA tubes from the retro-orbital sinus apoE.KO or apoE.KO/TRX0 transgenic mice. Plasma samples were separated by centrifugation at 630xg for 20 min at 4°C and frozen in 0.5 ml aliquots at -80°C until tested. For quantification of IL-6, IL-33 and MCP-1, we used the Millipore kit (reference

, Determination of anti-oxLDL antibodies -Lipoprotein isolation, Luminex 200 Millipore apparatus and xPONENT 3.1 software (Millipore

, 053 g/ml) were isolated from freshly drawn blood from healthy normolipidemic volunteers as previously described, dialyzed against PBS supplemented with 0.01% EDTA to prevent oxidation, sterilized by filtration and stored at 4°C under nitrogen. The relative electrophoretic mobility of LDL was evaluated on Hydragel

, Oxidation was initiated by incubation at 37°C with 5 µM CuSO 4 for 24 h. Oxidation was stopped by adding 20 µM EDTA. Native and oxLDL were screened for lipopolysaccharide (LPS) contamination by using a limulus amoebocyte lysate assay (Sigma), EDTA was removed by extensive dialysis of the LDL solution against EDTA free PBS, vol.163

, Before each assay, 96-well microtitration plates were freshly coated with 100 ?l of oxLDL (5 ?g/ml) in PBS overnight at 4°C. The wells were blocked with 1% bovine serum albumin for 2 h at room temperature. A 100 ?l aliquot of diluted sera (1:40) from each group of mice was added in quadruplicate wells and incubated for 2 h at room temperature. After three washes with PBS containing 0, vol.1

. Coulter, ) was added to each well, and the incubation continued for 2 h at room temperature. Plates were washed again, and the alkaline peroxidase activity was determined using orthophenylenediamine dichloride (OPD, Sigma) as a substrate and detected at 492 nm. TRX1 and TRX80 signal pathways

, Murine peritoneal macrophages were isolated from C57Bl/6, cultured at 0.5 x 10 6 cells/ml and untreated or treated with TRX1 (1 to 5 g/ml) or TRX80 (1 to 5 g/ml), PI3K inhibitor, Akt inhibitor, mTOR inhibitor, or a combination thereof. Expressions of macrophage M1 markers, MCP-1, TNF-, IL-6 and IL-1 and macrophage M2 markers, IL-10 and CD204 were investigated at the transcription levels using real-time polymerase chain reaction, Phosphorylation of Akt, pp.70-76

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