anti Notch intracellular domain, anti insulin degrading enzyme N terminal, anti neprilysin, anti apolipoprotein E, anti cAMP response element binding protein and anti phosphorylated CREB, anti Sp1 and anti B actin. After three washings with T TBS, membranes were incubated with IRDye 800CW or IRDye 680CW labeled secondary antibodies at 22 C for 1 h. Signals were developed selleckchem with Odyssey Infrared Imaging Systems. B actin was always used as internal loading control. Real time quantitative RT PCR amplification RNA was extracted and purified using the RNeasy mini kit, as previously described. Briefly, 1 ug of total RNA was used to synthesize cDNA in a 20 uL reaction using the RT2 First Strand Kit RT PCR. Mouse BACE 1, PS1, nicastrin, APH 1 and Pen 2 genes were amplified by using the corresponding primers designed and synthesized by Super Array Bioscience.
B actin was always used as an internal control gene to normalize for the amount of RNA. Real time PCR was performed in an Eppendorf ep realplex thermal cycler. Two microliters Inhibitors,Modulators,Libraries of cDNA was added to 25 uL of SYBR Green PCR Master Mix. Each sample was run in duplicate, and analysis of relative gene expression was done by using the 2 Ct method. Briefly, the relative change in gene ex pression was Inhibitors,Modulators,Libraries calculated by subtracting the threshold cycle of the target genes from the internal control gene. Based on the fact that the amount of cDNA doubles in each PCR cycle, the final fold change in gene expression was calculated by using the for mula relative Inhibitors,Modulators,Libraries change2 Ct.
Cell cultures N2A cells stably expressing human APP carrying the K670NM671L Swedish mutation were grown in Dulbeccos modified Eagle medium supplemented with 10% fetal bovine serum, 100 UmL penicillin, 100 ugmL streptomycin, Inhibitors,Modulators,Libraries and 400 ugmL G418, at 37 C in the presence of 5% CO2 as previously described. For each experiment, equal numbers of cells were pla ted in six well plates. 24 h later media were removed and fresh media containing either MK 591 or vehicle were added. After incubation for 24 h, supernatants were collected for AB and LDH measurement, and cell pellets harvested in lytic buffer for immunoblot analyses as described in the previous paragraphs. For transfection studies, N2A APPswe cells were transfected with 1 ug Myc tagged mE Notch 1 com plementary DNA overnight by using Lipofectamine 2000. The media were removed and fresh media containing MK 591, L685,458 Inhibitors,Modulators,Libraries or vehicle were added.
selleck After incubation for 24 h, cells lysates were collected NICD expression levels assayed by western blot analysis. Data analysis Data analyses were performed using SigmaStat for Windows version 3. 00. Statistical comparisons were performed by unpaired Students t test or the Mann Whitney rank sum test when a normal distribution could not be assumed. Values represent meanstandard error of the mean. Significance was set at P 0. 05.