Ic fibroblasts were followed RAAS System using a mouse embryo, day E13.5 like. The mouse embryos were cut and crushed in the presence of trypsin, and the cells washed with serum-free DMEM and 10% in the medium f Tales bovine serum DMEM. The wild-type and null MEF TG2 of the same passage were used in this experiment. HEK293 cells overexpressing TG2 and TG2 activity t mutant were prepared as described above. All cells were cultured in DMEM containing 10% FBS and 100 units / ml penicillin and streptomycin. To determine the doxorubicin-induced cell death and activation of TG2, all cells were mixed with 0.1 g / ml doxorubicin treated. To mediators of doxorubicin induced activation TG2 identified, the cells were for 1 h with 1 mM N-acetylcysteine, 20 M BAPTAAM, 2 mM EGTA, 30 g / ml of neutralizing antibody Rpern transforming growth factor, 10 pretreated g / ml, not like receptor 2 neutralizing antibodies body or 0 20 mM caffeine before doxorubicin treatment. Trypan blue exclusion analysis The cultured cells were harvested by centrifugation and resuspended in 500 l of DMEM. Since dead cells floating in the medium and the cells that remained on plates were collected. After the addition of trypan blue-L Solution were found Rbten cells using a H Mozytometers gez Hlt.
The percentage of dead cells was applied to the total number of cells. Assay the activity t in in situ TG TG activity t in situ was biotinamidopentylamine by measuring the amount of 5 in the proteins Installed will be tested. Both floating and attached cells were incubated together for 1 h with 1 mM BP and were harvested by centrifugation. Cell extracts were prepared by sonication followed by centrifugation. Cell extracts in coating buffer was added to each well of a 96 well microtiter plate. BP products were probed with streptavidin-horseradish peroxidase conjugate, followed by reaction with dihydrochloride ophenylenediamine. The absorbance at 490 nm was measured using a microplate spectrophotometer. Western blot analysis, the cells were in buffer containing 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1% Triton X-100 and a protease inhibitor cocktail lysed and centrifuged at 14,000 g for 10 min at4, the protein concentration of the supernatant using the BCA method. Each sample was separated by SDS-PAGE and transferred to nitrocellulose membranes.
After treatment for 1 h with 5% skim milk in Tris-buffered saline Solution, the membranes were separated with antique Incubated body against TG2, caspase 3 and actin for 2 hours. The membranes were subsequently End washed, incubated with HRP-conjugated secondary Rantik Body and using a chemiluminescent substrate L Solution, specified by the manufacturer. For visualization of TG activity t BP proteins in situ Formed with HRP-conjugated streptavidin followed by chemiluminescence detection have been explored. The statistical analysis of differences between two variables were analyzed by an unpaired Student t-test , and the differences between variables using ANOVA with Tukey’s multiple comparison. Differences were considered significant if the p value was 0.05. All statistical calculations were performed using Prism 4.0. RESULTS AFTER.