For experiments in hypoxia, plates were placed in a closed humidified chamber a

Phenylmethanesulfonyl fluoride, sodium orthovanadate, sodium fluoride, and a protease inhibitor mixture were purchased from Roche Molecular Biochemicals. Animal experiment Care, use, and treatment of animals were AS703026 supplier done in agree ment with the guidelines established by the Seoul National University Institutional Animal Care and Use Committee. Male BALBc mice were housed for 1 week before the experiments and maintained on a 12 h lightdark cycle, with food and water freely available. The mice were divided into the control and the treatment group. The treatment group mice were injected intraperitoneally with forskolin, and the control mice received an equal volume of Dulbeccos Phosphate Buffered Saline. After 6 h, the mice were exposed to whole body ray irradiation.<br><br> Expression constructs and transient transfection H1299 cells were 価格 AZD1152-HQPA transfected with a EE tagged constitu tively active mutant of long form stimulatory subunit of G protein in a pcDNA3 vector using the calcium phos phate method. A glutamine residue that is essential for the intrinsic GTPase activity is replaced with leucine in GsQL. A dominant negative mutant of PKA was a gift from Dr. G. Stanley McKnight. Constitutively active mutant of I kappa B kinase alpha S176ES180E and beta S177E181E were gifts from Dr. Dae Myung Jue. Small interfering RNAs against ATM were pur chased from Santa Cruz Biotechnology, and siRNA against PP2A B56 from Qiagen were purchased from Bioneer. siRNAs were trans fected using Lipofectaimine, and the cells were treated with other reagents at 48 h after transfection.<br><br> Preparation of cytosolic and nuclear fractions The cultured cells were harvested and then disrupted in lysis buffer A, 1 mM MgCl2, 0. 1% Triton X 100, protease inhibitor cocktail, and PMSF.The cell AMN-107 溶解度 lysates were centri fuged for 5 min at 800 g, and the supernatants were col lected to use as the cytosolic fractions. The resulting pellets were resuspended in lysis buffer B, PIC, and PMSF and centrifuged for 5 min at 20,000 g. The supernatants were collected to use as the nuclear fractions. Western blot analysis Western blotting was performed as previously described. Antibodies against Gs, Ku70, ATM, COX 1, phos phorylated cAMP response element binding protein, PP2A B56, IκB, p50 and p65 of NF κB were obtained from Santa Cruz Biotechnology.<br><br> Antibodies against Rad50, p ATM, H2AX, Ku80, CREB, DNA PKcs, poly polymerase, cleaved caspase 3, p AKT, AKT, p IκB, and Myc tag were obtained from Cell Signaling Technology. An anti body against B actin was purchased from Sigma, and an antibody against EE tag was purchased from Covance. An antibody against phosphorylated B56 of protein phosphatase 2A was kindly provided by Dr. Paul Greengard. The proteins were visualized using the Enhanced Chemiluminescence reagent and de tected using an LAS 3000. The densities of the protein bands were quantified using the Multi Gauge v2. 3 software, and the relative band densities were expressed as ratios of the corresponding control densities. Immunofluorescence microscopy H1299 cells were plated in 60 mm dishes and incubated until they became 60% confluent. The cells were trans fected with vector or GsQL plasmids, and after 24 h, they were irradiated with rays from a cesium irradiator. After 30 min, the cells were fixed with 4% paraformaldehyde for 20 min and permeated with 0. 5% Triton X 100 for 10 min.