The resulting supernatants were subjected to immunoprecipitation and immunoblot analysis. In Vitro Dephosphorylation Assay. 0.05 (Students test). Open in a separate windowpane Fig. S2. H&E staining of cross-sections prepared from your midcolon of 20-wk-old and 0.05 (ANOVA and Tukeys test). (= 15) and = 17) mice were treated with antibiotics from 4 wk of age, and disease activity for colitis was identified at 10, 15, and 20 wk of age. Data are Rabbit Polyclonal to KLRC1 from one representative experiment (and and and and mRNA, like that of mRNA, was previously found to be highest in the intestine (18, 25), whereas Eps8 is definitely indicated ubiquitously (27), we pursued the further characterization of CEACAM20 like a potential substrate for SAP-1 in the intestinal epithelium. Open in a separate windowpane Fig. S5. Lack of effect of SAP-1 ablation on tyrosine phosphorylation of SFKs in microvillus membrane portion. (section and subjected to immunoprecipitation and immunoblot analysis with the indicated antibodies. (and and and and and and 0.01, *** 0.001 (ANOVA and Tukeys test). ( 0.05, *** 0.001 (ANOVA and Tukeys test). (mRNA and then expressed relative to the normalized value for for 5 min after removal of cells debris. Isolation of RNA and Quantitative RT-PCR Analysis. Isolation of total RNA and quantitative RT-PCR analysis were performed as explained previously (44), with small modifications. In brief, total RNA was prepared from freshly isolated whole colon or colonic epithelial cells with the use of Sepasol RNA I (Nacalai Tesque) and an RNeasy Mini Kit (Qiagen), and first-strand cDNA was synthesized from 0.8 g of isolated total RNA with the use of a QuantiTect Reverse Transcription Kit (Qiagen). The cDNA fragments of interest were amplified either with the use of a QuantiTect SYBR Green PCR Kit (Qiagen) and ABI Prism 7700 sequence detection system (Applied Biosystems) or with Fast Start SYBR Green Expert (Roche) and a LightCycler 480 (Roche). The amplification was analyzed with the use of either SDS 1.7 software (Applied Biosystems) or LightCycler 480 software (Roche), and the abundance of each target mRNA was normalized by that of mRNA. Primer sequences (ahead and reverse, respectively) were as follows: IL-1, 5-CAACCAACAAGTGATATTCTACATG-3 and 5-GATCCACACTCTCCAGCTGCA-3; IL-4, 5-GGTCTCAACCCCCAGCTAGT-3 and 5-GCCGATGATCTCTCTCAAGTGAT-3; IL-6, 5-TAGTCCTTCCTACCCCAATTTCC-3 and 5-TTGGTCCTTAGCCACTCCTTC-3; IL-12 p40, 5-TGGTTTGCCATCGTTTTGCTG-3 and 5-ACAGGTGAGGTTCACTGTTTCT-3; IL-13, 5-CTCACTGGCTCTGGGCTTCA-3 and 5-CTCATTAGAAGGGGCCGTGG-3; IL-17, 5-TTTAACTCCCTTGGCGCAAAA-3 and 5-CTTTCCCTCCGCATTGACAC-3; TNF-, 5-CCCTCACACTCAGATCATCTTCT-3 and 5-GCTACGACGTGGGCTACAG-3; IFN-, 5-ATGAACGCTACACACTGCATC-3 and 5-CCATCCTTTTGCCAGTTCCTC-3; KC, 5-CAAGAACATCCAGAGCTTGAAGGT-3 and 5-GTGGCTATGACTTCGGTTTGG-3; MIP-2, 5-GGGCGGTCAAAAAGTTTGC-3 and 5-TGTTCAGTATCTTTTGGATGATTTTCTG-3; and GAPDH, 5-AGGTCGGTGTGAACGGATTTG-3 and 5-TGTAGACCATGTAGTTGAGGTCA-3. BrdU Incorporation Assay. Whole-body irradiation and a BrdU incorporation assay were performed as explained previously (40), with small modifications. In brief, adult mice were subjected (or not) to x-irradiation (total of 10 Gy). At 3.5 d after irradiation, the animals were injected i.p. with BrdU (50 mg/kg) and killed 2 h later on. The ileum or colon was fixed with 4% (wt/vol) paraformaldehyde, transferred to a series of sucrose solutions in 0.1 M sodium phosphate buffer, inlayed in OCT compound, and rapidly frozen in liquid nitrogen. Sections having a thickness of 5 m were incubated for 30 min at SR-2211 65 C with 0.025 M HCl, washed with 0.1 M borate buffer (pH 8.5), and exposed consecutively to pAbs to BrdU and secondary antibodies labeled with Cy3. Fluorescence images were acquired having a fluorescence microscope or a confocal laser-scanning microscope. BrdU-positive cells in 12 or 40 SR-2211 crypts per animal were counted with the use of ImageJ software (NIH), and the average quantity per crypt was identified. In Situ Closed-Loop System. Paracellular permeability of the colonic epithelium was identified as explained previously (45), with small modifications. In brief, mice were anesthetized and the colon was revealed by an abdominal midline incision. A 2-cm closed loop of the colon was prepared by ligation having a silk suture after washing the lumen of the gut. FD4 (10 mg/kg) in PBS was then injected into the loop, and peripheral blood was collected at 0, 15, 30, 60, and 120 min thereafter and centrifuged at 27,000 for 5 min to obtain the plasma portion. The fluorescence intensity of FD4 in the plasma samples was measured with the use of a fluorescence spectrophotometer (MTP-600F; Corona Electric). Preparation of Microvillus Membranes. Microvillus membranes were prepared from intestinal mucosal scrapings of adult mice as explained previously (46). In brief, SR-2211 the entire small SR-2211 intestine was eliminated, flushed, and opened longitudinally. The mucosal villi were then scraped off with glass slides and homogenized having a Polytron disrupter (PT-2100; Kinematica AG) for 3 min at 4 C in a solution comprising 50 mM.