e, duodenum, jejunum and ileum. Cubilin expression in the duodenum had not been pre viously reported. Calcitriol chemical structure To substantiate this, anti cubilin im munoblot analysis of intestinal extracts from wild type mice was performed. Similar to what has been observed in rat and canine ileal extracts, mouse ileum and Inhibitors,Modulators,Libraries jejunum extracts contained 460 kDa monomer, multimer and 200 kDa forms of cubilin. Cubilin was also detected in extracts of the duodenum, however the stoichiometry of the various immunoreac tive forms in the duodenum was different from that of ileum and jejunum. The cubilin membrane anchor, amnionless, was detected in all three segments of the small intestine. The data indicates that multiple amnionless polypeptides are apparent in extracts of the kidney and intestine and that their stoi chiometry differs between the two tissues.
Amnionless polypeptides have been previously described as having Mr values of 35 50 kDa, which corresponds to the range of polypeptides we observe in the intestine and kidney. The basis for these different forms is not clear, but may represent different posttranslational or postendocytic modifications of the protein as has been Inhibitors,Modulators,Libraries speculated. The cubilin ligand intrinsic factor was Inhibitors,Modulators,Libraries also observed in all three segments of the small intestine. Megalin expression in the small intestine was also examined and highest levels were detected in the ileum and jejunum and lowest levels in the duodenum. In all three segments of the small intestine, cubilin EGFP fluorescence was found in patches of intestinal villi, with regions of relatively strong fluorescence inter spersed in areas of little or no fluorescence.
Closer Inhibitors,Modulators,Libraries examination revealed that cubilin EGFP fluorescence was in discrete segments of each villus. To test the hypothesis that cubilin is monoallelically expressed Inhibitors,Modulators,Libraries in the small intestine, we performed qPCR analysis on microdissected villi from the ileum of Cubn del exon 1 6.EGFP mice that were predominantly ei ther EGFP positive, EGFP negative or both. If cubilin ex pression were biallelic, then the mosaic pattern might be explained by there being two populations of cells, one with both alleles inactive and the other with both alleles active. If this were the case then EGFP positive cells would express cubilin and EGFP mRNAs at equivalent levels. Furthermore, the EGFP negative cells would express neither transcript.
However, as shown in Figure 3I, predominantly EGFP negative cells were not only found to express cubilin mRNA but the levels were also significantly elevated as compared to the predominantly EGFP positive popula tion. Conversely, EGFP negative enterocytes expressed significantly lower EGFP mRNA levels as compared to EGFP positive cells. Based on these selleck Ruxolitinib findings the cubilin gene appears to be largely expressed from one allele in intestinal cells. We next examined sections of small intestine from Cubn del exon 1 6.