FIG. 5.

JNK activation is required for Daxx-mediated apoptosis. (A) JNK activation was monitored in L929-EV and L929-ΔDaxx cells by measuring the levels of phosphorylated JNK at the indicated times postinfection with rM51R-M virus or posttreatment with anisomycin. Phosphorylated JNK was detected via flow cytometry using antibodies specific for JNK phosphorylated at Thr183 and Tyr185. Data are expressed as the geometric mean fluorescence intensity (gMFI) compared to cells labeled with total rabbit serum as a primary antibody control (C). (B) Levels of phosphorylated JNK determined in A were normalized to levels of total JNK. Results are shown as the change (n-fold) in phosphorylated JNK in rM51R-M virus-infected cells and anisomycin-treated cells above the background observed in untreated cells. (C) Apoptosis in the presence and absence of a specific inhibitor of JNK activity (SP600125; 25 μM) was measured via flow cytometry by staining with annexin V and 7-AAD. Data are presented as percentages of viable cells (annexin V negative) and apoptotic cells (annexin V positive) and represent the averages ± SEM of three experiments (*P < 0.05 relative to samples not treated with JNK inhibitor).