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Fig. 2. Unlike CED-9, Bcl-2 cannot inhibit CED-4-dependent yeast death. (A) Yeast were transformed and spotted as described in the legend to Fig. 1. The ability of Bcl-2 to inhibit Bax-dependent and CED-4-dependent yeast death was tested, using Bcl-2 expressed from either the same promoter as CED-9 (ADH) or a very strong galactose-inducible promoter (GALL). The resulting expression levels were visualised by anti-Bcl-2 immunoblotting. Immunoblotting was also performed to monitor expression of GFP-tagged CED-4 and FLAG-tagged CED-9. An asterisk indicates a crossreacting yeast protein recognised by the anti-GFP antibody. Coomassie staining indicates protein loading. (B) Lowering expression of CED-4 using a methionine-repressible promoter (MET) yielded only weak CED-4-dependent yeast death upon co-expression with CED-3. Bcl-2 expression, directed by the intermediate-strength (ADH) or very strong (GALL) promoters, could not suppress even this weak death stimulus. (C) The two-hybrid yeast strain HF7c was transformed with the indicated plasmids or empty vector controls. Transformants were spotted onto minimal medium either containing histidine (growth confirms the presence of the plasmids) or lacking histidine (growth indicates interaction between the prey and bait proteins).
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