Ab183459

Whole-cell extracts were prepared in RIPA buffer containing protease inhibitor cocktail (Roche). The total protein concentration was determined with DC Protein Assay (Bio-Rad Laboratories). Equivalent doses of proteins were then size-fractionated in 10% SDS-PAGE, transferred to a 0.45 μm pore size PVDF Immobilon-P membrane (Millipore), subjected to 5% BSA blockage, and then incubated with the following antibodies: TLR4 (#ab183459, Abcam), RANK (#ab200369, Abcam), TRAF6 (#ab33915, Abcam), and β-actin (#sc-81178, Santa Cruz Biotechnologies. The bound antibodies were detected by the respective horseradish peroxidase-conjugated anti-IgG antibody (Amersham Biosciences) followed by ECL detection system (Amersham Biosciences) according to the manufacturer's instructions.

To further verify the changes of gene expression of PRRs signaling pathway, we measured the protein levels of Toll-like receptor-4 (TLR4), nucleotide-binding oligomerization domain protein-1 (NOD1), G-protein coupled receptor-43 (GPR43) and nuclear factor-κB (NF-κB) by using western blot analysis as described previously [27 (link)]. Total protein was extracted from the colonic mucosa by Tissue Protein Extraction Reagent (78,510, Thermo, USA), and expressions of those indexes were detected using primary antibodies against NF-κB p65 (ab140751, 1:1000; Abcam, UK), TLR4 (ab183459, 1:1000; Abcam), NOD1 (ab97278, 1: 1500; Abcam), GPR43 (ab131003, 1:1000; Abcam) and β-actin (SC-47778, 1:1500; Santa Cruz, USA) and Goat anti-Rabbit IgG (H + L) secondary antibody (31210, 1:5000; Thermo, USA). The quantitative data from western blot bands were expressed as the target protein OD/β-actin OD ratio. Furthermore, colonic mucin-4 (MUC-4) expression was measured by using immunohistochemistry with the primary antibody for MUC-4 (bs-1994R, 1:400; Bioss, USA) and HRP-conjugated secondary antibody (Thermo, USA). The detailed methods have been described previously [28 (link)]. The integral optical density (IOD) of each specimen was analyzed by Image Pro Plus 5.0.2. Each sample was used to prepare four slides, and each slide had four sections.