Cells were treated with Nogo P peptide for the indicated period of time at C

The cells were grown at 37 C in moist 5% CO2 atmosphere, and the method was regularly changed every 2 d. The mediwere changed with serum free medi12 h just before drug therapy. The cells were then treated with Abetor Abetfor 24 h. Epo at different concentrations were added to the cultures 1 h before the 24 h Abetexposure. 20 uM LY294002 Gefitinib EGFR inhibitor were added in to the cultures 1 h ahead of the Epo treatment. Analysis of cell viability Cell viability was assessed by MTT assay. Fleetingly, PC12 cells were seeded in 96 well culture plates at density of just one 104 cells per well. Following the cure of Abeta, Abeta, Epo or LY294002, the cells were subjected to the assay as previously described. Hoechst 33258 staining For Hoechst 33258 staining, cells were fixed with 401(k) par aformaldehyde. Cell nuclei were stained with Organism fluorescent dye Hoechst 33258 at final con centration of 5 ugml in PBS, for 20 min at room temperture in dim chamber, and then seen in fluorescence microscope and photographed. Western blotting The Western blotting analysis procedure was conducted as previously described. After the treatment, cells were washed twice with cold phosphate buffered saline and lysed on ice with cell lysis buffer, 60 ugmL aprotinin, 10 ugmL leupeptin, 1 ugmL pepstatin for 30 mininutes. The soluble fraction was obtained by centrifu gation at 14000 g for 20 min at 4 C. The attention of the protein was dependant on the BCassay. Equal amounts of the pro tein were divided in a 8 10 percent SDS polyacrylmide gel, the fixed proteins were electrotransferred onto PVDF or nitrocellulose membranes. The walls were subsequently blocked with 512-byte nonfat milk in TBST for 1 h at room temperature and incubated with PARP at 4, 1,5000 for betactin, 1,1000 for Cleaved caspase 3 and appropriate levels of primary antibody C overnight. The XL 888 membranes were then washed three times with TBST and probed with the corresponding secondary anti-bodies conjugated with HRP at room-temperature for 1 h. After washing, the signs were developed utilizing the ECL High level Wes tern Blotting Detection kit. Band intensi connections were quantified by densitometric analysis by utilizing an AxioCam electronic camerand the KS400 image analysis program. Research Datare expressed as mean standard deviation and were analyzed using SPSS 11. 0 mathematical software. Each method was per formed in duplicate in 3 5 independent experiments. Statistical analyses were performed using one way ANOVA, followed closely by the two tailed Students t test. Multiple comparison tests were used when appropri ate, and statistical significance was assumed at P 0. 05. Results Aftereffects of Abeton cell viability and cell apoptosis based on Hoechst and MTT 33258 staining respectively The MTT assay was used to ascertain the aftereffect of 20 uM Abeton the viability of the PC12 cell cul tures.