Figure 9. Diseased TAK1^ΔM Mice Exhibit Genomic Instability and TAK1 is regulated during mitosis.

(A) TAK1^ΔM mice were euthanized because of morbidity, and body and spleen weights were measured. Bone marrow was harvested, and short-term cultures were collected and processed for G-banding using standard methods. Analysis of 11–22 metaphase cells per mouse was performed, and the results listed indicated chromosomal deletions, additions, or breaks. (B–C) Primary mouse BMMs (B) or TAK1-WT MEFs (C) were treated with nocodazole (Noco, 100 ng/ml) for 14 h. Mitotic cells were collected by the mitotic shake-off method and released into fresh medium for the indicated times. Cell lysates were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. (D) TAK1-KO MEFs stably expressing FLAG-TAK1 were treated with nocodazole (100 ng/ml) for 14 h. Mitotic cells were collected by the mitotic shake-off method and released into fresh medium for the indicated times in the absence or presence of MG132 (25 µM; added 30 min after nocodazole release). Cell lysates were subjected to SDS-PAGE and immunoblotted with the indicated antibodies (bottom panels). Protein lysates were immunoprecipitated with anti-FLAG and immunoblotted with anti-ubiquitin (Ub) as indicated (top panel). The membrane was stripped and reprobed with anti-FLAG (middle panel). (E) TAK1-KO MEFs stably expressing FLAG-TAK1 were left untreated (A; asynchronized) or treated with nocodazole (100 ng/ml) for 14 h. Mitotic cells were collected by the mitotic shake-off method and released into fresh medium for the indicated times. Cells lysates were subjected to SDS-PAGE and immunoblotted with the indicated antibodies (right panels). Protein lysates were immunoprecipitated with anti-FLAG and an in vitro kinase assay with kinase-dead MKK6 (MKK6-kd) as the substrate was performed. The amount of total protein was adjusted so that an equivalent amount of TAK1 was immunoprecipitated from each time. The samples were subjected to SDS-PAGE, transferred to a membrane, and exposed to X-ray film (upper left panel). The membrane was stained with Ponceau S (bottom left panel) and probed with anti-TAK1 (middle left panel). Incorporation of ³²P into the substrate and TAK1 was quantitated using a PhosphoImager and presented as percent activation (graph). Asych, asynchronized.