Western Blot Analysis of Signaling Molecules

Extracted samples were separated by SDS–polyacrylamide gel electrophoresis (4% to 12%) and transferred to nitrocellulose membranes (GE Healthcare, Cardiff, United Kingdom). For analysis of signaling intermediates, membranes were blocked using Odyssey blocking buffer (30% in PBS, Li-Cor, Cambridge, United Kingdom) or milk Tris-buffered saline (5% milk, Tris-buffered saline, 0.1% Tween). TILRR was detected by an affinity-purified specific nonblocking rabbit peptide anti-TILRR antibody, as previously (1:1,000, custom made, Eurogentec) 13 (link), 14 (link). Incubation with antibodies against IL-1RI (1:500, Abcam, Cambridge, United Kingdom), tumor necrosis factor receptor 1 (TNFRI) (1:250, Abcam), TLR2 (1:1,000, Novus Biologicals, Abingdon, United Kingdom), TLR4 (1:1,000, Novus Biologicals), inhibitor kappa B alpha (IκBα) (1:1,000, Santa Cruz Biotechnology, Heidelberg, Germany), phospho-IκBα (1:1,000, Cell Signaling Technology, Leiden, the Netherlands), and β-actin (1:1,000, Santa Cruz) was followed by probing with a relevant secondary antibody and developed using LI-COR Odyssey (Li-COR, Biosciences, Lincoln, Nebraska) and quantitation using ImageJ (64-bit Java) (National Institutes of Health, Bethesda, Maryland).