Involvement of PC-PLC in LPS-induced JNK activation.

(A) PC-PLC inhibitor affects SEK/JNK activation; quiescent BAC-1.2F5 cells were pretreated with the PC-PLC inhibitor D609 (10 μmol/L, 60 minutes) before stimulation with 1.5 μg/mL LPS for 15 minutes. The presence of phosphorylated SEK and JNK was detected by immunoblotting with the corresponding antibodies. The inhibitor did not have any effect on the basal level of kinase phosphorylation. An anti-JNK1 blot is shown as loading control. (B) LPS stimulates PC-PLC; quiescent BAC-1.2F5 cells were stimulated with 1.5 μg/mL LPS for different times before solubilization. PC-PLC activity was determined in whole cell extracts as described in “Materials and methods.” Results are expressed as the percentage increase with respect to the control values. The plot in B represents the mean of 3 independent experiments, and vertical bars represent the standard errors of the mean. (C) Bacterial PC-PLC activates JNK in macrophages; quiescent BAC-1.2F5 cells were stimulated with 50 U/mL bacterial PC-PLC for 60 minutes before solubilization. The presence of phosphorylated JNK was detected by immunoblotting with the corresponding antibodies. An anti-JNK1 blot is shown as a loading control. (D) BIM pretreatment, but not down-regulation of DAG-dependent PKC, affects the activation of PC-PLC by LPS. Quiescent BAC-1.2F5 cells were left untreated (black bars) or were treated with BIM (10 μmol/L, 60 minutes; gray bars); alternatively, down-regulation of DAG-dependent PKC was performed as described in Figure 3B (white bars). Cells were stimulated with LPS for 15 minutes before lysis and determination of PC-PLC activity in whole cell extracts. Results are expressed as the percentage increase with respect to the control values.