Sustained activation of JNK signaling in S2 cells lacking Relish activation. (A) S2 cells were treated with dsRNAs specific to kenny, ird5, or relish for 3 d and then unstimulated or stimulated with LPS for the length of time indicated. Cell lysates were prepared and analyzed by immunoblotting with antibodies specific to the phosphorylated forms of JNK. (B) S2 cells were treated with 0.1% (v/v) dimethylsulfoxide (DMSO), 1 μg/mL actinomycin D (Act D), or 25 μg/mL cycloheximide (CHX) for 1 h and then unstimulated or stimulated with LPS for the length of time indicated. Cell lysates were prepared and analyzed as in A with antibodies specific to the phosphorylated forms of JNK (Phos-JNK) and Relish. For Relish, only the full-length (FL) form is shown. (C) S2 cells were mock-treated or were treated with ird5, kenny, or dredd dsRNAs and analyzed as in Figure 2B.