For immunolocalization of TLR4/MD2, confocal images were obtained (Radiance system 2100; Bio-Rad Labs, Hercules, CA) and for immunolocalization selleck inhibitor of occludin fluorescent images were obtained with an Olympus Provis AX70 microscope (Olympus, Melville, NY); images were analyzed with Scion Image software (Scion, Frederick, MD). Measurement of phosphorylated myosin light chain by Western blot. Phosphorylated myosin light chain (p-MLC) expression was measured by Western blot of ileum as previously described (17). Briefly, 30 g of proteins were used, samples were loaded in precast 7% Tris acetate gel, and the gel was run for 60 min at 125 volts (Invitrogen Power Case 500). The proteins were transferred (1 h at 30 V, 220 mA) from the gel to a Fluorotrans polyvinylidene difluoride membrane (Pall, East Hills, NY).
Skim milk (10%) in TBS-Tween 20 (TBST) was used to block. Primary antibody dilution was 10 ��l in 10% milk-TBST [glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) (14C10) rabbit monoclonal antibody, no. 2118 (Cell Signaling, Beverly, MA); p-MLC (18Thr)-R, no. sc-19848-R (Santa Cruz Biotechnoloy)]. Milk-TBST (10 ��l in 25%) of secondary antibody [anti-rabbit IgG horseradish peroxidase-linked, no. 7074 (Cell Signaling), were used as well as horseradish peroxidase (1:2,000; Cell Signaling). The membrane was exposed to film for 30 min and developed in a 100 Plus automatic X-Ray film Processor (All Pro Imaging, Hicksville, NJ). The film was analyzed by Imagequant version 5.1 software (Amersham, Biosciences, Amersham, UK) (17).
Quantitative assessment of gut microbiota order abundance by sequence analysis of the microbial 16S rRNA gene. The materials and methods are described in detail [see supplemental information and Ref. 20 (Supplemental data for this article may be found on the American Journal of Physiology: Gastrointestinal and Liver Physiology website.)]. Cecal contents were removed and maintained on dry ice for same-day processing by phenol-chloroform-sodium acetate-ethanol- and bead beating-based cellular lysis and nucleic acid isolation/purification methods. Each quantitative PCR (qPCR) well was run in triplicate and contained 10 ��l of 2�� Takara Perfect Real Time master mix (7.2 ��l of water, 0.
8 ��l of a 10 ��M F/R primer mix, 2 ��l of either an optimized dilution of 1:500 of extracted template DNA in DNase/RNase-free water for specimen analysis or a serial dilution series of Carfilzomib bacterial reference genomic DNA for standard curves); all reactions were paralleled by a nontemplate water control analysis. Cycling conditions were as follows: 95��C for 20 s; 40 repeats of the following steps: 95��C for 4 s, 30 s annealing. SYBR green fluorescence was detected with a Bio-Rad Chromo4 Real Time PCR Detector on a Dyad Disciple Peltier Thermal Cycler. Melting curves were obtained from 55 to 90��C, with fluorescence measurements taken at every 1��C increase in temperature.