FIG. 7.
(A) Ectopic Bcl-2 expression blocks BAD cleavage in IL-3-starved 32Dc13 cells. Parental (lanes 1 and 2) and Bcl-2-expressing (lanes 3 and 4) 32Dc13 cells were infected with pLXSP-HA WT BAD; after selection in puromycin (2.5 μg/ml), transduced cells (2 × 105/ml) expressing high levels of murine BAD were deprived of IL-3 for 8 h (lanes 2 and 4) and harvested. Cell extracts were subjected to an SDS–4 to 15% gradient polyacrylamide gel, transferred to a nitrocellulose membrane, and blotted with the anti-C-terminus (C-20) BAD antibody. (B) Ectopic Bcl-2 expression does not block BAD cleavage in death ligand-treated Jurkat T cells. Parental (lanes 1 to 4) and Bcl-2-expressing (lanes 5 to 8) Jurkat cells were infected with pLXSP WT hBAD; after selection in puromycin (2.5 μg/ml), transduced cells (2 × 105/ml) expressing high levels of hBAD were treated for 6 h with anti-FAS antibody (lanes 2 and 6), TNF-α (lanes 3 and 7), and TRAIL (lanes 4 and 8) and harvested. Cell extracts were subjected to an SDS–4 to 15% gradient polyacrylamide gel, transferred to a nitrocellulose membrane, and blotted with polyclonal anti-BAD serum (H-168). Arrows indicate cleaved BAD.