Modifications of full-length inducible caspase 9. (A) The full-length inducible caspase 9 molecule (F'-F-C-Casp9) consists of 2 FK506 binding proteins (FKBPs) linked with a Ser-Gly-Gly-Gly-Ser linker to the small and large subunit of the caspase molecule. The amino acid sequence of one of the FKBPs (F') is codon-wobbled to prevent homologous recombination when expressed in a retrovirus. F'F-C-Casp9_C→S contains a cysteine to serine mutation at position 287 that disrupts its activation site. In constructs F'F-Casp9, F-C-Casp9, and F'-Casp9, either the caspase activation domain (CARD), one FKBP, or both, were deleted respectively. All constructs were cloned into MSCV.IRES.GFP as EcoRI-XhoI fragments. 293T cells were transfected with each of these constructs and 48 hours after transduction expression of the marker gene GFP was analyzed by flow cytometry. In addition, 24 hours after transfection, 293T cells were incubated overnight with 100 nM CID and subsequently stained with the apoptosis marker annexin V. The mean and standard deviation of transgene expression level (mean GFP) and number of apoptotic cells before and after exposure to CID (% annexin V within GFP⁺ cells) from 4 separate experiments are shown. (B) Coexpression of the inducible caspase 9 constructs of the expected size with the marker gene GFP in transfected 293T cells was demonstrated by Western blot using a caspase 9 antibody specific for amino acid residues 299-318, present both in the full-length and truncated caspase molecules as well as a GFP-specific antibody. Additional smaller size bands likely represent degradation products. Degradation products for the F'F-C-Casp9 and F'F-Casp9 constructs may not be detected due to a lower expression level of these constructs as a result of their basal activity. Equal loading was confirmed by blotting for actin.