The left panel shows magnified the eGFP channel only

t was cut 19320832 into small pieces and washed a number of times in ice cold PBS. The volume equivalent of heart tissue was measured and then homogenized with 2 times the tissue volume of Mitochondrial Isolation Buffer and in presence of protease inhibitor cocktail, using 20534345 quick pulses in a Waring blender. The lysate was centrifuged twice at 3,0006g at 4uC for 20 minutes to remove nuclei and unbroken cells. The supernatant was then centrifuged at 10,0006g for 20 minutes at 4uC. The supernatant was kept for cytosol preparation while the pellet was washed with 500 mL of MIB buffer and centrifuged again at 10,0006g for 20 minutes at 4uC. The mitochondrial pellet was finally re-suspended in a minimum volume of MIB buffer and the protein concentration was determined by Bradford assay. For the preparation of cytosol, the first mitochondrial supernatant fraction was subjected to further centrifugation steps at 4 degrees Celsius in order to remove remaining intracellular organelles. The supernatants containing cytosolic proteins were snap frozen in liquid nitrogen. Prior to use in the assay, cytosols were thawed and subjected to centrifugation at 180,0006g and the protein concentrations were determined. Reconstitution of MDV formation in vitro Five milligrams of purified cow heart mitochondria were washed 3 times in 1 mL of MIB by centrifugation at 10,0006g at 4uC. The first wash was performed in presence of protease inhibitor cocktail. The washed mitochondria were then incubated in the budding reaction in 500 ml of an osmotically controlled, buffered environment including an energy regenerating system, where the final concentrations of reagents were: 220 mM mannitol, 68 mM sucrose, 80 mM KCl, 0.5 mM EGTA, 2 mM magnesium acetate, 20 mM Hepes pH 7.4, 1 mM ATP, 5 mM Succinate, 80 mM ADP, 2 mM K2HPO4, pH 7.4. Volumes were controlled using concentrated stock solutions to ensure a controlled osmolarity in each reaction. Following the reaction, the intact mitochondria were removed from the mixture by two sequential centrifugations at 10,0006g at 4C. The supernatants containing the MDV fraction were treated with 0.5 mg/ml trypsin for 10 minutes at 4C, unless prepared for the sucrose density centrifugation. Following trypsin treatment, loading buffer was added and the samples were 300817-68-9 cost separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted. We tested the effect of a panel of damage by adding to the assay either: 50 mM Antimycin A, 50 mg/ml chloramphenicol, 100 mM GTPcS, 125 mM oligomycin, 200 mM xanthine and 0.4U xanthine oxidase, 20 mM NEM, all from Sigma. Materials and Methods Ethics Statement Fresh bovine heart was obtained with permission from a government-licensed abattoir in St. Albert, Ontario. 2 Biochemical Reconstitution of MDVs Sucrose gradient fractionation All steps were carried out at 4uC. The supernatants obtained from budding reactions were adjusted to 40% in mitochondrial isolation buffer and loaded on the bottom of a discontinuous sucrose gradient with steps at 40%, 30%, 20%, and buffer. Samples were subjected to centrifugation at 150,0006g for 6 hours and fractions were collected for analysis as indicated. postfixed in 1% osmium tetroxide in 0.1 M Na cacodylate buffer, en bloc stained in 3% aqueous uranyl acetate, dehydrated in ascending ethanol and embedding in Spurr epoxy resin and processed as previously described. Results Reconstitution of MDV formation in vitro To determine the mechanism of MDVs formation and cargo incorpor