Figure 2.

Cdk inhibitor flavopiridol selectively inhibits Bcl-2 modification in cells arrested by antimicrotubule drugs. Bcl-2-overexpressing cells were treated with antimicrotubule drugs alone or in combination with various kinase inhibitors (1 µM). After ∼1 day, cell lysates were prepared, normalized for either cell equivalents of total protein content, and subjected to SDS-PAGE/immunoblot analysis using anti-Bcl-2 antibodies with ECL-based detection. Dark and open arrowheads indicate unmodified and modified Bcl-2, respectively. In (A), detergent-solubilized lysates (2x10⁶ cells/sample) were prepared from 293-Bcl-2 cells that were cultured without treatment (-), ∼2 µM nocodazole (+) or the combination of nocodazole and Cdk-inhibitor flavopiridol, MEK inhibitor PD098059, or p38 kinase inhibitor SB203580. In (B), 697-Bcl-2 cells were treated with nocodazole alone or in combination with flavopiridol as indicated. In (C), 697-Bcl-2 cells were cultured with 300 nM paclitaxel, 1 µM PKC-inhibitor GF109203X, or combinations of these reagents, followed by preparation of lysates (normalized for total protein content) and SDS-PAGE/immunoblot analysis of Bcl-2.