Coexposure to Src inhibitors and UCN-01 leads to unphosphorylated BimEL accumulation, enhanced p34^cdc2activation, and increased γH2A.X expression. (A) MM cell lines were exposed (24 hours) to UCN-01 (U266, 150nM; RPMI 8226, MM.1S, and MM.1R, 100nM) ± 1μM BMS354825, after which phosphorylation and expression of BimEL were assessed by Western blot analysis using a Bim antibody (Calbiochem) that is able to distinguish both the phosphorylated (p, slowly migrating) form from the unphosphorylated (up, fast-migrating) form. Vertical lines have been inserted to indicate repositioned gel lanes. (B) After being treated with UCN-01 (U266, 150nM; RPMI 8226, MM.1S, and MM.1R, 100nM) ± SKI606 (U266 and RPMI 8226, 2μM; MM.1S and MM.1R, 1μM), Western blot analysis was performed to examine dephosphorylation/activation of p34^cdc2 at the inhibitory site Y15. In parallel, total p34^cdc2 was monitored for comparison. (C) Cells were exposed (24 hours) to UCN-01 (U266, 150nM; RPMI 8226, MM.1S, and MM.1R, 100nM) ± the indicated concentrations of BMS354825, after which phosphorylation of the atypical histone H2A.X at S139 (designated γH2A.X) was monitored by Western blot analysis. (D) MM.1S and U266 cells were treated as described in (B) and (C) for 24 or 48 hours, respectively, after which Western blot analysis was performed to monitor PARP cleavage. (E) Primary CD138⁺ MM cells (Pt2) and their CD138⁻ counterparts were exposed (24 hours) to 100nM UCN-01 ± the indicated concentration of BMS354825, after which expression of Bim using an antibody (ProSci) that recognizes total protein levels of 3 isoforms (EL, L, and S), T14/Y15 phosphorylated p34^cdc2, γH2A.X, and PARP were assessed by Western blot analysis. CF indicates cleavage fragment.