Enforced activation of MEK1/2 by B-Raf disables R115777 but not PD184352 to block ERK1/2 activation and to potentiate γH2A.X expression mediated by Chk1 inhibitors. (A) U266 cells were stably transfected with a construct encoding full-length B-Raf, and WB was performed to monitor B-Raf expression as well as resulting MEK1/2 phosphorylation (inset). Blots were subsequently stripped and reprobed using β-actin antibodies to ensure equivalent loading and transfer of proteins. Cells were then exposed to 150 nM UCN-01 or 2 μM Chk1i with or without 5 μM PD184352 or 5 μM R115777 for 48 hours, after which the percentage of annexin V⁺ cells was determined by flow cytometry. The results represent the means plus or minus SD for 3 separate experiments performed in triplicate. **P < .01; NS indicates no significance (P > .05). (B) Alternatively, after 24-hour exposure to drugs, WB was performed to monitor expression of phospho-ERK1/2 and γH2A.X. Results are representative of 3 separate experiments.