Figure 2.
Mcl-1 and Bcl-xL are necessary and sufficient for cytosolic inhibitory activity. (A) Mcl-1 and Bcl-xL correlate with inhibitory activity after ammonium sulfate fractionation. Ammonium sulfate was added to 1 mL of S100 (5 mg/mL) up to 30% (lanes 3,4) or 50% (lanes 5,6). Following incubation at 4°C for 1 h, the supernatant (Sup) and pellet (Pel) were separated by centrifugation (20,000g). The pellet was resuspended in 1 mL of Buffer A. Both the supernatants and pellets were dialyzed in Buffer A overnight. All fractions were assayed for inhibitory activity (as described in Materials and Methods) and evaluated for Mcl-1 and Bcl-xL. (B) HeLa S100 (36 mg) was first precipitated with 30% ammonium sulfate. The resulting pellet (7.5 mg) was resuspended and dialyzed in Buffer A and then loaded onto a 1-mL Hi-trap Q Sepharose column (Amersham) equilibrated in Buffer A. The protein was eluted with a gradient from 0 to 750 mM NaCl (in Buffer A) over 14 mL. Inhibitory activity was assayed for buffer alone (lane 1), input (lane 2), Q flow through (lane 3), and fractions eluting from Q sepharose (lanes 4-13). The amount of Mcl-1 and Bcl-xL in each sample and the mitochondria (lane 1) was measured by Western blot. (C) HeLa S100 was immunodepleted as described in Materials and Methods. Inhibitory activity was assayed in buffer alone (lane 1), S100 mock-immunodepleted (lane 2), depleted of Mcl-1 (lane 3), Bcl-xL (lane 4), or both (lane 5). The amount of Mcl-1 and Bcl-xL was determined by Western blot for each S100 sample. (D) Recombinant Mcl-1 (rMcl-1) and Bcl-xL (rBcl-xL) were prepared as described in Materials and Methods. S100, rMcl-1, and rBcl-xL were analyzed for inhibitory activity. The levels of Mcl-1 and Bcl-xL in recombinant fractions were compared with those in S100. Mitochondria solubilized in Buffer A with 1% NP-40 were analyzed for Mcl-1 and Bcl-xL to compare the levels of these proteins in mitochondria to those in the fractions.