An ABI PRISM 7900 HT sequence detection system was used for monitoring the level of SYBR green fluorescence. A dissociation curve was produced at the end of the cycling phase to ensure a single PCR product and no primer dimer. Amplification efficiencies were determined for all genes by serial dilution of cDNA using E 10. We checked that a slope may between 3. 2 and 3. 5 was obtained for each primer couple to calculate the relative expression levels of the selected genes by using the 2 Ct method. The gene encoding Ribosomal Protein L32 was chosen as the internal reference for each sample. The mock infected sample at T0 of each replicate was used as a calibrator for all samples of one replicate. The mean and the standard deviations for each Inhibitors,Modulators,Libraries time of each replicate were calculated.

Flow cytometry The Inhibitors,Modulators,Libraries anti porcine MHC class I monoclonal antibody PT85A and the anti porcine MHC class II mAb MSA3 were purchased from VMRD. The mAb HOPC 1 was used as con trol Ab. PE conjugated goat Abs to mouse IgG2a were pur chased from Southern Biotech. PK15 cells from three kinetics Inhibitors,Modulators,Libraries replicates, either just after mock infection or infection with the GFP expressing PrV Inhibitors,Modulators,Libraries strain and 8 h after mock infection or pi were trypsinized, resuspended in pig serum for 20 minutes at 4 C, washed in D PBS and resuspended in FACS buffer. Cells were incubated with 50 l of diluted primary antibody for 30 minutes at 4 C and then, after washing, in 50 l of diluted PE conjugated goat anti mouse IgG2a for 30 minutes at 4 C. After two washes in FACS Buffer, the cells were fixed in BD CellFix solution and analyzed using a FACScalibur flow cytometer.

Background The 70 kDa ribosomal protein S6 kinase is a mitogen activated serine threonine kinase that has a criti cal role in control of cell cycle, growth and survival. p70S6K is encoded by RPS6KB1, which is located at 17q23 and is amplified and overexpressed in 10 30% of breast cancer cell lines and primary breast cancers. The overexpression of p70S6K is associated with aggres Inhibitors,Modulators,Libraries sive disease and poor prognosis of breast cancer patients. p70S6 kinase is located downstream of PI3K AKT mTOR pathway, which is activated by HER2 receptors, insulin like growth factor receptor and estrogen receptor in breast cancer. p70S6K itself is activated by 3 phos phoinositide dependent protein kinase 1 and mammalian target of rapamycin kinase.

p70S6 kinase regulates protein synthesis by activating 40S ribos omal protein S6, leading to an increased rate of transla tion of the class of 5TOP mRNA transcripts. These transcripts encode critical com ponents of the cellular translational machinery, thus pro moting protein synthesis. e-book Additionally, p70S6K has a crucial role in cell growth by regulating cell size and pro gression of cell cycle. Recently, p70S6K has been reported to inactivate the pro apoptotic molecule BAD by phosphorylation, thereby also promoting cell survival.