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. http://game-rapidshare.com/Vandewalker-grandma-of-Eichstedt-from-Playa?Lindseymo=247
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Tissue and cell lysis and fractionation

Freshly harvested tissues (tissue: buffer ratio, 1:10 w/v) were homogenized in RIPA buffer (cat# 9806, Cell Signaling, Dallas TX), centrifuged at 10,000 g, and supernatants used as heart tissue lysates. Sub-organelle fractions were simultaneously prepared by following the published protocol [23]. Briefly, tissues were suspended in homogenization buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM DTT, 25 ?g/ml spermine, 25 ?g/ml spermidine, and protease inhibitor cocktail) containing 250 mM sucrose; tissue: buffer ratio, 1:20) and homogenized at 4°C by using a dounce homogenizer. The homogenates were centrifuged at 800 g for 15 min at 4o C, and supernatants and pellet collected. The pellets were re-solubilized in four volumes of homogenization buffer containing 2 M sucrose by repeated pipetting, homogenized with a single stroke of the dounce homogenizer, filtered to remove debris, layered on top of 4 ml cushion of homogenization buffer/2M sucrose, and centrifuged for 35 min in a swing-bucket ultracentrifuge at 80,000 g. After aspirating the supernatant, pellets containing pure nuclei were stored at -70°C. The supernatants from low speed centrifugation step were centrifuged at 6000 g for 15 min, and resultant pellets and supernatants were stored as mitochondrial and cytosolic fractions, respectively. Markers of nuclear (Lamin B), and mitochondrial (COIV subunit) fractions were evaluated in all mitochondrial fractions, and mitochondrial fractions that exhibited > 6% of contaminant were re-centrifuged as described above to ensure purity. The mitochondrial fractions were also confirmed for purity by PCR evaluation of nuDNA encoded GAPDH fragment and mtDNA encoded 7S fragment (S1B Fig).

Cardiomyocyte or HeLa cells were seeded in 6-well plates (1x106 cells per well), and incubated with T. cruzi (cell/parasite ratio of 1:5) in presence or absence of 1 ?M PJ34 for 24 h. Cells were suspended in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 2.5 mM KH2PO4, and 1 mM Na3VO4), and incubated on ice for 30 min. Cell lysates were centrifuged at 12,000 g at 4°C for 15 min and the resultant supernatants were stored at ?80°C. For fractionation, cells (7?106/ml) were incubated on ice for 30 minutes in buffer A (10 mM HEPES, pH 7.9, 10 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF) containing 0.625% NP-40 and 1% protease inhibitor cocktail. Cell lysates were centrifuged at 4°C at 10,000 g for 1 min and supernatants were stored as a cytosolic fraction. Pellets were washed with buffer A containing 1.7 M sucrose, re-suspended in buffer B (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF), and centrifuged at 4°C at 13,000 g for 5 minutes. The resultant supernatants were stored at ?80°C as nuclear extracts. Mitochondria were isolated from cells as above.

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