Ff26a f9

Antibodies for flow cytometric detection of surface antigens: α2-integrin (P1E6-C5), α3-integrin (ASC-1), α4-integrin (9F10), α5-integrin (NKI-SAM-1), α6-integrin (GoH3), F4/80 (BM8, BioLegend); β1-integrin (sc-53711, Santa Cruz); CRT (ab2907), ERp57 (ab10287, Abcam). Antibodies for flow cytometric detection of intracellular antigens: CRT (ab2907), PDI (ab2792) and cytochrome C (6H2.B4, Biolegend). Antibodies for immunoprecipitation and immunoblotting: α4-integrin (HP2/1), CRT (PA3-900, ThermoFisher), ERp57 (ab10287, Abcam), and GAPDH (FF26A/F9, BioLegend). Antibodies for integrin activation and phagocytosis assays: β1-integrin (9EG7) and CD47 (B6H12, BD Biosciences).

Cells from monocyte subsets (sorted by FACS) were lysed in RIPA buffer (Sigma) containing 1 × protease inhibitors (Roche). Lysates were loaded onto 10% SDS-polyacrylamide gels (3 × 10⁵ cells per lane). After transfer, the membranes were probed with anti-RelA, dilution 1:200, [532301] (#MAB5078; R&D Systems) or anti-GAPDH, dilution 1:2000, [FF26A/F9] (#649201; Biolegend), followed by HRP-conjugated anti-mouse secondary antibody, dilution 1:5000, (#W4021, Promega). Detection was performed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the protein band sizes were quantified using Image J, normalized to GAPDH.

Peripheral blood samples from 47 early arthritis patients with specific homozygous genotypes for the SNPs of interest were used to analyse Gal1 expression in lymphocytes. First peripheral blood mononuclear cells were isolated by density gradient (Biocoll Separating Solution; Biochrom). Then, the monocytes were purified by positive selection using CD14 MicroBeads and autoMACS (both from Miltenyi Biotec) following the manufacturer’s instructions. T lymphocyte enriched fractions were lysed at 4 °C (during 30 min) in Tris-buffered saline (50 mM Tris-Cl, pH 7.5, 150 mM NaCl) 1% NP40 with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Whole lysates were analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membranes and probed with the Human Galectin-1 antibody (R&D Systems) and the anti-GAPDH antibody FF26A/F9 (BioLegend, San Diego, CA, USA) in Tris buffered saline–Tween 20. Bound antibodies were conjugated with horseradish peroxidase secondary antibodies, and membranes were developed by enhanced chemiluminescence with Super-Signal West Femto chemiluminescent substrate (Pierce Chemical, Dallas, TX, USA). Densitometric analyses were performed with ImageGauge 3.46 software (Fujifilm, Greenwood, SC, USA).