This was probed with end labeled 32P labeled oligonucleotides in perfectHyb buffer at 37 C and washed using low and medium stringency conditions. All probes used are listed in Additional file Enzastaurin Phase 3 2 Table S6. Small RNA molecular analyses For enzymatic analyses of RNA material, assays were done as previously published. Briefly, either 10 ug or 50 ug small RNA enriched RNA sample was spiked with a control sample. For the Terminator assay, the sample mixture was treated with Terminator enzyme, following the Inhibitors,Modulators,Libraries provided protocol from the manufacturer. For the capping assay, the ScriptCap m7G capping system was used with the alternate cap zero capping protocol. After enzymatic treatment, samples were phenol chloroform extracted and resolved on a 12% polyacrylamide gel.
Northern blot analysis was performed using a radiolabeled probe to detect the small RNA of interest. Strand specific RT PCR analyses We used SuperScript III first strand synthesis kit for strand specific cDNA synthesis and PCR analysis. E. histolytica HM Inhibitors,Modulators,Libraries 1 IMSS total RNA was trea ted with DNase I, and purified strand specific primer for each gene was added to 0. 5 ug total RNA reaction and heated to 65 C for 5 min. The tem perature was lowered to 55 C, and prewarmed cDNA syn thesis mix was added to the reaction and incubated at 55 C for 50 min. The reaction was terminated at 85 C for 5 min, chilled on ice, and 1 ul of RNase H was added to each tube and incubated for 20 min at 37 C before proceeding to PCR. In each primer reaction, both RT and RT reactions were performed, and the final cDNA volume was 20 ul.
PCR was performed using 1 ul cDNA for a 30 ul PCR reaction for 33 cycles. Half of the volume of PCR reaction was loaded Inhibitors,Modulators,Libraries on a gel for visualization. Background Cubilin is a 460 kDa peripheral membrane glycoprotein, anchored to the plasma membrane via an amino ter minal amphipathic helix and through interactions with the transmembrane protein, amnionless, forming the so called cubam complex. Cubilin is expressed by the absorptive epithelia of tissues such as renal prox imal convoluted tubules, ileum, and yolk sac, where it mediates the endocytosis of numerous ligands, in some cases acting in concert with another endocytic receptor, LRP 2/megalin. Cubilin was first described as the intrinsic factor cobalamin/vitamin B12 receptor important for intestinal absorption of vitamin B12.
Mutations of the cubilin gene are the cause of Imerslund GrAsbeck syndrome, also known as selective vitamin B12 malabsorption with proteinuria. Proteinuria Inhibitors,Modulators,Libraries in these individuals results from the inability Inhibitors,Modulators,Libraries of the cubilin deficient Cabozantinib solubility kidney to reabsorb ligands that filter across the glomerulus, in cluding albumin and apolipoprotein A I, the major apo lipoprotein of HDL. In vitro studies have shown that cubilin mRNA ex pression is stimulated by retinoic acid and that it is not sterol regulated.