Figure 4. PKD1 is essential for SR-induced activation of MAPKs and NF-κB and subsequent expression and production of cytokines and chemokines in vitro.
AMJ2-C11 and MLE12 cells were transiently transfected with 100 nM of non-target siRNA (NT siRNA; control) or PKD1-specific siRNA (PKD1-siRNA; PKD1-knockdown) using lipofectamine. (A) Messenger RNA levels of the indicated genes were analyzed by RT-PCR. (B) PKD1 protein levels of the indicated genes were examined using Western blot analysis. (C-E) Control or PKD1-knockdown cells were stimulated with medium, SR-Ag, or IFNγ for 45 min (Panel C), 2 h (for IFNγ in Panel D), 4 h (Panel D), or 24 h (Panel E). Activation status of PKD1, MAPKs, STAT1, and NF-κB (assessed by degradation of IκBα and IκBβ) was detected by Western blot (Panel C). Messenger mRNA levels of the indicated cytokines and chemokines were analyzed by RT-PCR (Panel D). Levels of cytokines and chemokines in culture supernatants were analyzed by ELISA (TNF-α, IFNγ, IL-6, IL-12, MIP-2) or multiplex sandwich assay (KC, MCP-1) (Panel E). Data represent the mean concentration (pg/ml) ± SD of triplicates. Actin and GAPDH were used as loading controls. All experiments were done three times with similar results.