ERK1/2 phosphorylation is increased by SMI-4a treatment. (A) 6812/2 cells were incubated with 10μM SMI-4a in the absence of serum for 24 hours and extracts of these cells subjected to Western blotting with antibodies to p-ERK1/2 (Thr202/Tyr204), total ERK, and GAPDH. (B) Jurkat cells were incubated with the indicated concentrations of SMI-4a in the serum-free media for 8 hours. Extracts were then probed as in panel A. (C) 6812/2 cells were incubated with varying amounts of SMI-4a and U0126 in serum-free media for 24 hours. Viable cells were then identified by trypan blue exclusion and the percentage of survival compared with the untreated control was plotted. (D) Based on data in panel C, Chou and Talalay's combination index for mutually exclusive agents was constructed across combined doses for SMI-4a and U0126. Estimated indices (●) and corresponding 95% credible intervals (horizontal lines) are shown. The combination index is not estimable for situations in which the kill rates are 100%. Therefore, no index is provided for any combination in which at least one dose was 40μM. (E) 6812/2 cells were treated with DMSO as a control or 10μM SMI-4a with or without 10μM U0126 for 14 hours. Extracts were probed as described in panel A. (F) The spleen and thymus studied in Figure 4D were subjected to Western blotting with antibodies to phospho-ERK1/2, total ERK, and β-actin, as a control.