Ab131003

The total protein from each caecal mucosa sample was extracted with RIPA Lysis Buffer (cat.SN338, Sunshine Biotechnology Co., China). The concentration of the total protein was measured by the Pierce™ BCA Protein assay kit (cat.23225, Themo Fisher, USA). The detection metod of cadidate protein was indicated in the previous study [19 (link)]. The primary antibodies used in the current study included TLR4 (sc-293,072, Santa Cruz, USA), GPR41 (ab103718, Abcam, USA), GPR43 (ab131003, Abcam, USA), NF-κB (An365, Beyotime, China), p38 (ab31828, Abcam, USA), ERK1/2 (ab17942, Abcam, USA), β-tubulin (KC-5 T01, Kang Chen Bio-tech, China), and GAPDH (AP0066, Bioworld, USA). The expression level of each candidate protein is shown as the fold change of the average value between the LC and HC groups.

Cells and tissue samples were lysed for protein extraction using RIPA assay. The concentration of each protein sample was determined by a BCA (bicinchoninic acid) kit. Equal amounts (50 mg) of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, United States). Membranes were blocked using 5% non-fat dry milk with TBST buffer for 1 h, then incubated overnight at 4° C with GPR43 (1:1000, ab131003, Abcam), MFN2 (1:500, ab205236, Abcam), PPARγ (1:1000, ab178860, Abcam), NOX-1 (1:1000, ab121009, Abcam), EBP50 (1:1000, ab3452, Abcam), p47phox (1:1000, ab166930, Abcam), NLRP3 (1:1000, 15101, Cell Signaling Technology, Danvers, MA, US), Caspase-1 (1:1000, sc-392736, Santa Cruz Biotechnology), IL-1β (1:1000, 12242, Cell Signaling Technology, Danvers, MA, US), and β-Actin (1:5000, sc-47778, Santa Cruz Biotechnology). After washing, membranes were probed further with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:5000, Santa Cruz Biotechnology). After washing with TBST for 15 min, immunoreactive bands were exposed by enhanced chemiluminescence method (Thermo Fisher Scientific, Waltham, MA, USA).

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.