Anti trif

Mφ transfected with either Mal siRNA or control siRNA were washed with PBS and disrupted with lysis buffer (50 mmol L⁻¹ Tris-HCl, 250 mmol L⁻¹ NaCl, 5 mmol L⁻¹ EDTA, 1 mmol L⁻¹ dithiothreitol, 5% glycerol, 0.2% Nonidet P-40 at pH 8) containing a protease inhibitor cocktail (Roche Applied Science). Protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA). Equal amounts of whole cell lysates (50–100 μg) were loaded in each lane for separation by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking in Tris-buffered saline containing Tween 20 (0.1%) (T-TBS) and milk (5%) at room temperature for 2 h, the membrane was then incubated at 4°C overnight with primary antibodies. After washing with T-TBS, the membrane was incubated at room temperature for 1 h with HRP-conjugated anti-rabbit IgG secondary antibody (Cell Signaling Technology, Danvers, MA). The primary anti-Mal (Abcam, Cambridge, UK), anti-MyD88 (Cell Signaling Technology), anti-TRIF (Novus Biologicals, Littleton, CO), anti-TLR-4 (Novus Biologicals) and anti-β-actin (Sigma- Aldrich) antibodies were diluted and used as described in the manufacturer’s instructions. The density of each band was quantified by densitometric analysis with Image J software (Image Processing and Analysis in Java) from the NIH (https://imagej.nih.gov/ij/).