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. 2012 Aug 23;7(8):e43970. doi: 10.1371/journal.pone.0043970

Figure 4. CpG DNA-mediated induction of Irak-m promoter activity is dependent on IRAK2 and PKD1 as well as IRAK4 and IRAK1.

Figure 4

Panels A, B, and D. RAW264.7 cells were transiently cotransfected with empty vector or plasmids encoding DN-IRAK4 (A), DN-IRAK1 (B), or DN-IRAK2 (D) and Irak-m-promoter-luciferase plus pRL-TK-luciferase reporters, NF-κB-luciferase plus pRL-TK-luciferase reporters, or AP-1-β-galactosidase reporter. Cells were stimulated with medium or CpG DNA (6 μg/ml). Luciferase activity in cell extracts was analyzed by the Dual-Luciferase Reporter Assay System and normalized using pRL-TK-luciferase activity in each sample. β-galactosidase activity in equal amounts of cell extracts was analyzed using the Galacto-Light Plus Reporter gene assay. Data are the mean relative light unit (fold induction from luciferase activity or β-galactosidase activity of the indicated reporter in the unstimulated cells) ± SD of triplicates. Significant differences from luciferase activity or β-galactosidase activity of the indicated reporter in the cells transfected with empty vector and stimulated with CpG DNA are indicated (* p<0.05; ** p<0.005). Panel C. Control luciferase-knockdown macrophages (Luc-shRNA) or Prkd1-knockdown macrophages (PKD-1shRNA) were cotransfected with Irak-m-promoter-luciferase and pRL-TK-luciferase. Transfected cells were treated with medium, CpG DNA (6 µg/ml), or LPS (50 ng/ml) for 36 hr. Luciferase activity in cell extracts was analyzed by the Dual-Luciferase Reporter Assay System and normalized using pRL-TK-luciferase activity in each sample. Data are the mean relative light unit (fold induction from luciferase activity of unstimulated cells) ± SD of triplicates. Significant differences from luciferase activity in Luc-shRNA cells stimulated with CpG DNA (** p<0.005) or LPS (## p<0.005) are indicated. All experiments were repeated at least three timeswith similar results.