Enhanced chemiluminescence detection method

Complement C6 and S100A9 proteins were assessed by Western blot. Briefly, 2 µL of serum was run on 4–12 % NUPAGE Bis–Tris polyacrylamide gels (Invitrogen), transferred and incubated with anti-C6 monoclonal antibody (dilution 1/2000; ab71942; Abcam) or with anti-S100A9 polyclonal antibody (dilution 1/1000; sc20173; Santa Cruz). We then incubated with a mouse or a rabbit secondary antibody (dilution 1/5000; Dako) to detect C6 or S100A9, respectively. Proteins were revealed with an enhanced chemiluminescence detection method according to the manufacturer’s instructions (GE Healthcare). Band intensities were quantified by ImageQuant LAS 4000 software (GE Healthcare) and are expressed as pixel counts representing integrated signal intensities.

Cells were washed with cold PBS 1X and scraped in RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium desoxycholate) supplemented with protease inhibitors (Roche Diagnostics, Meylan, France). Whole cell lysates were then sonicated and centrifuged at 10000 rpm for 10 min at 4 °C. Protein concentration was estimated using the Bradford protein assay (Bio-Rad, Marnes-la-Coquette, France). Total protein extracts (30 μg) were dissolved in Laemmli buffer (Bio-Rad) and separated by 15% SDS-PAGE. Proteins were transferred onto PVDF membranes (GE Healthcare, Amersham, UK) and non-specific binding was blocked in TBS-Tween 20 buffer (0.5 mM Tris-HCl, 45 mM NaCl, 0.05% Tween 20, pH 7.4) containing 5% nonfat milk. Membranes were incubated with the primary antibody anti-A-FABP (clone AB13979, 1:1000, Abcam, Paris, France). Protein blots were probed with anti-β-actin (clone AC-15, 1:40000, Sigma-Aldrich) as controls for protein loading. Bound primary antibody was detected using an HRP-conjugated secondary antibody anti-rabbit IgG (1:5000 or 1:10000) obtained from BD Biosciences (Le Pont de Claix, France). Proteins were visualised using an enhanced chemiluminescence detection method (GE Healthcare) followed by film exposure (Hyperfilm ECL, GE Healthcare), or by using ChemiDoc XRS+ with Image Lab Software (Bio-Rad).