Inhibitor kappa b alpha iκbα

Collected kidney tissues were homogenized in radioimmunoprecipitation assay buffer (RIPA; Sigma-Aldrich, USA) which contains 1% protease inhibitor cocktail (Sigma-Aldrich, USA) and 2% phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich, USA). Protein concentration was analyzed via Bradford method, and 30 µg samples were separated by using a 10% SDS-PAGE gel and transferred electrophoretically onto nitrocellulose membranes (0.45 m; Bio Basic, Inc., USA). After being blocked in 5% bull serum albumin (BSA) for 4 h at 4°C, the membranes were blotted at 4°C overnight with primary antibodies phosphor-nuclear factor-κB (P-NF-κB) receptor, total-NF-κB (T-NF-κB) (1 : 500; Santa Cruz, USA), inhibitor kappa B alpha (IκBα), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz, USA) at dilution of 1 : 500. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 2000; Santa Cruz, USA). Chemiluminescence was detected by using ECL detection kits (GE Healthcare, UK). The intensity of the bands was quantified by scanning densitometry using software Image J (National Institutes of Health, Bethesda, USA).

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).