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. 2005 Jun;25(11):4488–4500. doi: 10.1128/MCB.25.11.4488-4500.2005

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Copyright © 2005, American Society for Microbiology

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FIG. 2. — Bim induction in mitochondria by Gadd45a protein. (A) The HeLa Gadd45a-inducible cell line was grown in DMEM in the presence of tetracycline at a concentration of 2 μg/ml. After removal of the tetracycline, the cells were collected at the indicated time points for the preparation of cellular protein. A total of 100 μg of whole-cell protein was examined for activation of caspase-9, caspase-8, JNK, and p38 kinases. In addition, total JNK and p38 protein were also examined. (B) Mitochondrial protein was isolated from HeLa Gadd45a-inducible cells (see Materials and Methods) at different time points and used for the detection of Bim and Bax. (C) Cytochrome c release was measured in mitochondrion (Mit)-free cytosol protein after inducible expression of Gadd45a in Hthe removal of tetracycline, caspase-9 inhibitor (LEHD-fmk) or caspase-8 inhibitor (IETD-fmk) was added to the cell cultures. After 48 h, the cells were collected for the preparation of cellular protein. A total of 100 μg of whole-cell protein was examined for the activation of caspase-9, caspase-8, and PARP cleavage. Bim induction of mitochondria and cytochrome c release were examined as in panels B and C.

FIG. 2. — Bim induction in mitochondria by Gadd45a protein. (A) The HeLa Gadd45a-inducible cell line was grown in DMEM in the presence of tetracycline at a concentration of 2 μg/ml. After removal of the tetracycline, the cells were collected at the indicated time points for the preparation of cellular protein. A total of 100 μg of whole-cell protein was examined for activation of caspase-9, caspase-8, JNK, and p38 kinases. In addition, total JNK and p38 protein were also examined. (B) Mitochondrial protein was isolated from HeLa Gadd45a-inducible cells (see Materials and Methods) at different time points and used for the detection of Bim and Bax. (C) Cytochrome c release was measured in mitochondrion (Mit)-free cytosol protein after inducible expression of Gadd45a in Hthe removal of tetracycline, caspase-9 inhibitor (LEHD-fmk) or caspase-8 inhibitor (IETD-fmk) was added to the cell cultures. After 48 h, the cells were collected for the preparation of cellular protein. A total of 100 μg of whole-cell protein was examined for the activation of caspase-9, caspase-8, and PARP cleavage. Bim induction of mitochondria and cytochrome c release were examined as in panels B and C.