Amplification

Amplification Brefeldin was performed by using SYBR Premix Ex Taq II. The primers used for amplification included the following GAPDH mRNA levels were determined as an internal control. Electrophoretic mobility shift assays Nuclear extracts were prepared in hypertonic buffer. Double stranded oligonucleotide probes that were derived from the IBP gene promoter were incubated with the probe for 30 min at 30 C. The protein DNA complexes were resolved using non denaturing PAGE and were detected by autoradiography. For the cold probe compe tition assay, unlabelled probe was added to the nuclear protein extracts one hour before the detection was per formed. In the supershift Inhibitors,Modulators,Libraries assay, 1 ul of an anti p53 anti body was incubated with the nuclear extracts for 1 h at room temperature prior to the addition of the radiolabeled probe and the implementa tion of PAGE.

Chromatin immunoprecipitation assay The ChIP assays were performed using an EZ ChIPTM Chromatin Immunoprecipitation Kit following the manufacturers instructions. Briefly, cells were cross linked with 1% formaldehyde and a p53 antibody or control IgG, which was used to precipitate Inhibitors,Modulators,Libraries the crosslinked proteinchromatin. The DNA fragments were analysed using PCR with a primer set that was designed to amplify the ?305 to ?150 region of the IBP gene that harbours p53 binding site. Cell survival assays A cell survival assay Inhibitors,Modulators,Libraries was performed in triplicate with a Cell Counting Kit 8. The cells were seeded in 96 well plates at 5 103 cellswell 24 h before the cisplatin treatment.

The culture medium was then replaced with fresh medium that con tained different concentrations of cisplatin, which ranged from 0 to 32 ugml, and the cells were Inhibitors,Modulators,Libraries cultured in this medium Inhibitors,Modulators,Libraries for 24 h. Following the incubation, 10 ul of CCK 8 solution was added to each well, and after 1 h, the absorbance value of each well was read at 450 nm. The cell growth rate was calculated as the ratio of the absorbance of the experimental well to that of the blank well. The IC50 values were calculated. Annexin V PI flow cytometry assay Flow cytometry assay was performed by using Caliber II sorter and Cell Quest FACS system. Alexa fluor 647 conjugated Annexin V and PI was incubated for 15 min according to the manufacturers protocol. About 104 cells were mea sured per sample. Background Muscle mass is a primary determinant of muscle strength, and is strongly associated with the performance of activ ities of daily living and the level of independence of the elderly. The phosphatidylinositol 3 kinase Akt mammalian target of rapamycin pathway is recognized as a possible mechanism that regulates muscle mass. In mammals, skeletal muscle hypertrophy available occurs as a result of an increased size, instead of increased number, of preexisting skeletal muscle fibers.

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