Analyzing PRRs Signaling Pathway

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.