Ll lysis buffer shiny. Cell extracts Sorafenib 475207-59-1 were centrifuged and the whichever type Walls were collected. Caspase 3 activity was t measured by the producer 鈥 檚 protocol. Briefly, cell lysates were incubated with 75 ll of caspase-3 substrate 3 hours incubation at 378C in triplicate in 96-well plates, and then caspase 3 activity t was by using caspase-3 kit colorimetric detection Fairs the absorption of free p-nitroaniline generated by cleavage of Ac DEVD pNA colorimetric substrate. Western blot after stimulation with inhibitors cilostazol and other HT22 cells were scraped off and resuspended in lysis buffer containing 50 mM Tris-Cl, 150 mM NaCl, 0.02% sodium azide, 100 mg phenylmethylsulfonyl fluoride / ml, 1 mg / ml aprotinin and 1 % Triton X-100. To prepare nuclear extracts, the cells were lysed in buffer A. After incubation for 5 min at 48C, were collected nuclei by centrifugation at 4000 rpm, and the pellets were resuspended in buffer B and min on ice for 20 min. After centrifugation at 13,000 rpm for min 7 30 lg total protein of each sample on 10% SDS-polyacrylamide gels were loaded, and the separated proteins Were transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk and then incubated with a 1:1000 dilution of antibodies Rpern against p53, p53 phosphorylation on Ser15, GSK 3b, 3b phosphorylation at GSK Ser9, b catenin, b-catenin phosphorylation at Ser675, or phosphorylated CK2a CK2a. Antique Body against GSK 3b phosphorylation at Tyr216 was obtained from Upstate L Measurements. After incubation with horseradish peroxidase-conjugated spots-secondary Ren Antique Body IgG is, the intensity t of chemiluminescence using an image analyzer Fluorochem gel. The membrane was with an anti-b actin, which was used as controlled probed The internal. Hoechst 33342-F Evaluate the properties staining of apoptotic cells, Hoechst 33342-F has been posted to this coloring. The cultures were washed twice with PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 15 min and then incubated with light 10 lg / ml Hoechst 33342 in PBS at 378C for 15 minutes. Neurons were photographed with nuclei by fluorescence microscopy. The number of condensation nuclei was at least five different repr Sentative areas quantified for each treatment. Measuring the relative activity of t-CK2 CK2 activity t in lysates of HT22 cells in culture was loud using an assay kit CK2 CycLex manufacturer S instructions. Briefly, 100 ll reaction mixture was added to the wells and incubated for 30 min at 308C. After the wells were washed, 100 ll HRPconjugated detection antibody Body added to each well and incubated at room temperature for 60 min. After washing the wells were 100 ll reagent substrate tetramethylbenzidine, added to each well and incubated at room temperature for 10 15. Subsequently End was applied 100 LL 1 N H2SO4 to stop the reaction. CK2 activity t was measured by absorbance at 450 nm. Preparation of small interfering RNAs Maraviroc CCR5 inhibitor and transfection The siRNA oligonucleotide was synthesized by Bioneer CK2a. The duplex-controlled The negative control siRNA oligonucleotide was used as On. siRNA molecules were transfected into cells using a transfection reagent TransFast. The CK2a siRNA target sequences that were used in this report were s.