The immune complexes resulted were pel leted, washed three times with ice cold PBS, reconsti tuted with SDS sample buffer and then resolved on the SDS PAGE. Western blotting of lysates from GFP tagged selleck chemical TCTP overexpressing cells following eto poside treatment was performed by anti GFP and protein specific antibodies. Western blotting and im munoprecipitation of lysates from Flag tagged adNull and adTCTP infected cells following etoposide treat ment were performed by using anti Na,K ATPase 1, Apaf 1 and protein specific antibodies. Image of west ern blot was visualized and obtained using LAS 3000 image analysis system. In vitro activation of apoptosome formation To obtain the S 100 extract, HeLa cells were harvested through centrifugation. After washing the cells, cells were then resuspended Inhibitors,Modulators,Libraries in buffer, 1 mM EGTA and EDTA, 0.

1 mM phenylmethylsulfonyl fluoride, 10 ug ml leupeptin aprotinin, and Inhibitors,Modulators,Libraries 1 mM dithiothreitol. Then, reconstituted Inhibitors,Modulators,Libraries cells were homogenized with a Dounce glass homogenizer, and the resultant cell homogenates were subjected for centrifugation at 10,000 g for 10 min to extract the nuclear and mitochon drial organelles. The supernatants containing S 100 frac tion were obtained and were mixed with 1 mM dATP 10 uM cytochrome c at a 2. 5 mM Mg2 concentration. Where indicated, recombinant TCTP protein was supple mented in the reaction mixture. Isolation of cytosolic and mitochondrial fractions Following centrifugation, cells were harvested and the mitochondrial and cytosolic fractions were isolated using commercial kit according to the manufacturers instructions.

In brief, cells were incu bated with Reagent A for 2 min on ice and then trans Inhibitors,Modulators,Libraries ferred to Dounce homogenizer Inhibitors,Modulators,Libraries for homogenization. After adding the Reagent C, the mixtures were then centrifuged at 700 g for 10 min at 4 C. The super natant were then collected and further centrifuged at 3,000 g for 15 min at 4 C to pellet the mitochondria. The resulting supernatant was designated as cytosolic fraction and the mitochondrial precipitate was washed with Reagent C followed by centrifugation at 12,000 g for 5 min at 4 C. The purity of cytosolic and mitochon drial fractions was confirmed by the western blotting by detecting the immunoactivity of actin and COX IV, respectively. Measurement of cytochrome c release Cytochrome c release was measured by western blotting or quantified using a fluorescent dye.

Following the isolation of cytosolic and mitochondrial fractions from HeLa cells as described above, cytochrome c contents in each fraction were analyzed by immunoblot ana lysis using anti cytochrome c specific antibody. To quantify the cytochrome c re lease, cells were mixed with a buffer containing 20 mM HEPES, 10 mM KCl, 1. 5 mM MgCl2, 1 mM EGTA and EDTA, 1 mM AEBSF, 8 mM DTT, and selleck inhibitor 250 mM sucrose, supplemented with digitonin.