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. Author manuscript; available in PMC: 2011 Feb 3.
Published in final edited form as: J Biol Chem. 2004 Apr 28;279(28):28954–28960. doi: 10.1074/jbc.M403490200

FIG 2. Recombinant tBid induces the release of cytochrome-c and SMAC from isolated mitochondria and binds to Bcl-XL in vitro.

FIG 2

A, isolated HeLa mitochondria (10 µl, 1 mg/ml) were incubated with increasing amounts (0–133 nm in 50 µl) of tBid. The supernatant (S) and pellet (P) fractions were separated by centrifugation, resolved on SDS-PAGE, and analyzed by Western blotting with either cytochrome c (top) or SMAC (bottom) antibody. In B, Ni2+-NTA resin (10 µl) was incubated first with His-Bcl-XL(ΔC) (15 µg) and then with tBid (15 µg). The Ni2+-NTA-bound protein was eluted with 500 mm imidazole, and the samples were resolved by SDS-PAGE and stained with Coomassie Blue (lane 5). In the control experiment, tBid was directly incubated with the resin without preincubation with Bcl-XL(ΔC) (lane 4). Individual Bcl-XL(ΔC) monomers (24 kDa) are resolved in lane 2, and individual tBid monomers (15 kDa) are resolved in lane 3. Molecular weight markers are shown in lane 1.