Expression, kinase activity, and TCR rearrangements in p210^bcr/abl in the leukemic tissues ofBCR/ABL^tg/−p53^+/− mice.

For the expression assessment (A), 50 μg of protein aliquots extracted from the enlarged thymus or pleural effusion of 9BCR/ABL^tg/−p53^+/−leukemic mice (no. 1-9 on Table 1) was separated by 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, and probed with the anti-Abl monoclonal antibody AB3 (1:500). To determine kinase activity (B), 1 mg of protein aliquot extracted from the same tissues shown in Figure 3A was incubated with AB3 (1:200) and the immunoprecipitated proteins were subjected to in vitro kinase assays. Phosphorylated proteins were separated by 6% SDS-PAGE, dried, and autoradiographed. For both the expression and kinase activity assessments, the thymus of a normalBCR/ABL^−/− control mouse was the negative control, and the thymus of aBCR/ABL^tg/− leukemic mouse21was the positive control. The arrow indicates the position of p210^bcr/abl; the positions of the molecular markers are shown on the left. To describe TCR rearrangements (C), 5 μg of DNAs digested with EcoRI was separated by 0.7% agarose gel, blotted to a nylon membrane, and probed with a phosphorus 32-dCTP–labeled TCRγ probe. DNA extracted from normal thymus tissue was used as a control (denoted by C). Molecular markers are shown on the left.