Involvement of PKC in LPS-initiated SEK and JNK activation.

(A) The PKC inhibitor BIM inhibits SEK/JNK activation by LPS; quiescent BAC-1.2F5 cells were treated with the PKC inhibitor BIM (10 μmol/L, 60 minutes) before stimulation with 1.5 μg/mL LPS for 15 minutes. The presence of phosphorylated SEK/JNK and of unmodified JNK1 was detected with the corresponding antibodies. (B) Prolonged TPA treatment of BAC-1.2F5 cells down-regulates DAG-dependent PKCs; cells were either left untreated or incubated with TPA (5 μmol/L in DMSO) or with DMSO alone for 24 hours. PKC δ, ε, and ζ were detected by immunoblotting. (C) Down-regulation of DAG-dependent PKC does not affect SEK/JNK activation by LPS; down-regulation of DAG-dependent PKCs was performed as described in B before stimulation with 1.5 μg/mL LPS for 15 minutes, cell lysis, and immunodetection of phosphorylated SEK/JNK and of unmodified JNK1.