Bmf: A Proapoptotic BH3-Only Protein Regulated by Interaction with the Myosin V Actin Motor Complex, Activated by Anoikis

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The cDNA libraries from day 17 mouse embryos or from mouse embryos from embryonic day 9 to day 1 postpartum were prepared in pAD-GAL4-2.1 (HybriZAP-2.1 kit, Stratagene). The bait vector was made by cloning mouse mcl-1 lacking the sequences encoding its hydrophobic COOH-terminus into pGBT-9 (Clontech). Yeast transformation and plasmid rescue were performed as previously described (11). Of the 7 × 10⁵ clones screened, one positive clone was obtained. Interaction between Mcl-1 and the novel protein was confirmed by β-galactosidase staining (11). Sequence analysis revealed that the clone was a partial one lacking the 5′ end. This partial clone was used as the probe to isolate full-length clones by screening a cDNA library derived from the p53^–/– KO52DA20 thymoma cell line. Human bmf was isolated by screening a human, activated T cell cDNA library using mouse bmf as probe. To screen for Bmf-interacting proteins, mouse bmf was subcloned into a pGBT-9 derivative harboring the gene for chloramphenicol acetyltransferase as the selection marker. Out of 5 × 10⁶ clones screened, 60 positive clones were initially selected.

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Rat monoclonal antibodies (mAbs) to dynein light chains or Bmf were generated as described (27). Antibodies were purified either on a protein-G column (Amersham Pharmacia) or on a Sepharose column conjugated with MAR 18.5 antibody (monoclonal mouse secondary antibody to rat Igκ). Monoclonal antibody 11F7 (rat γ2a/κ) recognizes mouse and human DLC1 and DLC2, whereas 10D6 (rat μ/κ) detects mouse and human DLC1 but not DLC2 [Web fig. 2 (17)]. Monoclonal antibodies 9G10 and 12E10 (both rat γ2a/κ) detect endogenous mouse and human Bmf by Western blotting and immunoprecipitation. To generate polyclonal antibodies against Bmf, New Zealand White rabbits were immunized with recombinant mouse Bmf. Sera were purified over a Sepharose column conjugated with recombinant mouse Bmf protein.

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We thank L. O'Connor and K. Newton for help with experiments and sequence analysis, L. Cullen and S. Novakovic for expert technical assistance, J. Visvader and W. Alexander for reagents, and G. Filby for editorial assistance. We are grateful to J. Adams, P. Bouillet, A. Harris, and S. Cory for critical review of the manuscript. This work was supported by fellowships and grants from the National Health and Medical Research Council (Canberra; Reg. Key 973002), the U.S. National Cancer Institute (CA 80188), the Dr Josef Steiner Cancer Research Foundation, the Leukemia and Lymphoma Society of America, the Human Frontiers Science Program, and the Sylvia and Charles Viertel Charitable Foundation. The sequences for mouse bmf, human bmf, and mouse dlc2 have been deposited with GenBank (accession numbers:, and, respectively).