PMC

Panels A, B and D. RAW264.7 cells were transiently cotransfected with pGL3 basic luciferase (control vector) or Irak-m-promoter-luciferase and pRL-TK-luciferase reporters. (A) Cells were stimulated with medium, CpG DNA (6 μg/ml), or non-CpG DNA (6 μg/ml) for 24 hr. (B) Cells were stimulated with medium, CpG DNA (6 μg/ml), or non-CpG DNA (6 μg/ml) for the indicated time periods. (D) Cells were stimulated with medium, CpG DNA (the indicated concentration), or non-CpG DNA (the indicated concentration) for 24 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 (RLU; fold induction from luciferase activity in the unstimulated cells) ± SD of triplicates. Statistical differences from the unstimulated control are indicated (^* p<0.05; ^** p<0.005). Panel C. RAW264.7 cells were stimulated with medium or CpG DNA (6 μg/ml) for the indicated time periods. Messenger RNA levels of Irak-m and β-actin (loading control) were detected by RT-PCR. All experiments were done more than three times with similar results.

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.

RAW264.7 cells were transiently transfected with non-target siRNA (NT siRNA; control) or Irak2-siRNA (Irak2-knockdown) using lipofectamine. Panel A. Messenger RNA levels of the indicated genes were analyzed by RT-PCR. Panel B. Levels of the indicated proteins were analyzed using Western blot assay. Panels C – E. Control or Irak2-knockdown cells were stimulated with medium (M), CpG DNA (6 μg/ml; C), or IFNγ (25 ng/ml; I) for the indicated time periods. (C, top) The activation status of PKD1 and MAPKs was detected by phospho-specific Western blot assay. (C, bottom) Quantitation of panel C top by densitometry. The density of each protein band was quantitated by densitometry and normalized to the density of the actin band in the same sample. Data represent the fold induction from the normalized densitometric value of each protein band of the unstimulated NT-siRNA control sample. (D) To detect NF-κB binding activity to the Irak-m promoter region, a ChIP assay was performed with anti-p65 Ab or isotype control IgG. DNA bound to p65 Ab or IgG was purified and used as a template for PCR with the Irak-m promoter-specific primer set that detects the Irak-m promoter region containing the putative NF-κB (2) consensus site or the Irak-m-3′ end-specific primer set. Actin was used as a loading control. IP, immunoprecipitation. (E, top) Total RNA was extracted and RT-PCR for Irak-m was performed. Actin was used as a loading control. (E, bottom) Quantitation of panel E top by densitometry. The density of Irak-m mRNA band was quantitated by densitometry and normalized to the density of the actin band in the same sample. Data represent the fold induction from the normalized densitometric value of Irak-m mRNA band of the unstimulated NT-siRNA control sample. Data represent results obtained from three separate experiments.

RAW264.7 cells were cotransfected with Irak-m-promoter-luciferase plus pRL-TK-luciferase, NF-κB-luciferase plus pRL-TK-luciferase, or AP-1-β-galactosidase and empty vector or plasmids encoding DN-p38, DN-MEK1, or DN-JNK1. The transfected 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 cell extracts was analyzed using the Galacto-Light Plus Reporter gene assay. Equal concentrations of cell lysates were used for the AP-1-β-galactosidase 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. Statistical differences from luciferase activity or β-galactosidase activity of the indicated reporters in the cells transfected with empty vector and stimulated with CpG DNA are indicated (^* p<0.05; ^** p<0.005). Panel D. RAW264.7 cells were stimulated with medium, CpG DNA (6 μg/ml), or non-CpG DNA (6 μg/ml) for 1 hr. To detect AP-1 binding activity to the Irak-m promoter region, a ChIP assay was performed with anti-c-Jun Ab or isotype control IgG. DNA bound to c-Jun Ab or IgG was purified and used as a template for PCR with the Irak-m promoter-specific primer set that detects Irak-m promoter region containing a putative AP-1 binding consensus site or with the Irak-m-3′ end-specific primer set. Actin was used as a loading control. IP, immunoprecipitation. Panel E. RAW264.7 cells were stimulated with medium, CpG DNA (6 μg/ml), or non-CpG DNA (6 μg/ml) for 1 hr. To detect nuclear DNA binding activity of AP-1, equal amounts of nuclear extracts (3 μg/lane) were subjected to EMSA using ³²P-labeled double-stranded ODN containing the AP-1 binding consensus sequences in the Irak-m promoter region as a probe. Panel F. RAW264.7 cells were transiently cotransfected with wild-type or AP-1 (-821/-815) site-mutated (AP-1 mut) Irak-m-promoter-luciferase and pRL-TK-luciferase. Cells were stimulated with medium or CpG DNA (6 μg/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 represent the mean RLU (fold induction from luciferase activity of wild type Irak-m promoter-luciferase reporter in the unstimulated cells) ± SD of triplicates. Statistically significant differences from luciferase activity of wild type Irak-m promoter-luciferase reporter in the cells stimulated with CpG DNA are indicated (^** p<0.005). Panel G. RAW264.7 cells cotransfected with pRL-TK-luciferase plus Irak-m-promoter-luciferase, CREB-luciferase, or NF-κB-luciferase and empty vector or vector encoding DN-CREB 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. Data are the mean relative light unit (fold induction from luciferase activity of the indicated reporter in the unstimulated cells) ± SD of triplicates. Significant differences from luciferase 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 H. RAW264.7 cells were treated with medium, CpG DNA (6 μg/ml), or non-CpG DNA (6 μg/ml) for 1 hr and a ChIP assay was performed with anti-pCREB Ab or isotype control IgG. DNA bound to pCREB Ab or IgG was purified and used as a template for PCR using the Irak-m promoter-specific primer set that detects the Irak-m promoter region containing a putative CRE consensus site or with the Irak-m-3′ end-specific primer set. Actin was used as a loading control. IP, immunoprecipitation. Panel I. RAW264.7 cells were transiently cotransfected with full length or CRE (−139/−131) site-mutated (CREB mut) Irak-m-promoter-luciferase reporters and pRL-TK-luciferase and then stimulated with medium or CpG DNA (6 μg/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 represent the mean RLU (fold induction from luciferase activity of wild type Irak-m promoter-luciferase reporter in the unstimulated cells) ± SD of triplicates. Statistically significant differences from luciferase activity of wild type Irak-m promoter-luciferase reporter in the cells stimulated with CpG DNA are indicated (^* p<0.05). All experiments were repeated at least three times with similar results.

RAW264.7 cells were transiently cotransfected with pRL-TK-luciferase and full length (FL) or 5′-deletion mutant (Δ-1086, Δ-756, Δ-406, Δ-215, or Δ-49) Irak-m promoter-luciferase reporters and then stimulated with medium, CpG (6 μg/ml), or IFNγ (25 ng/ml) for 24 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 represent the mean RLU (fold induction from luciferase activity of wild type Irak-m promoter-luciferase reporter in the unstimulated cells) ± SD of triplicates. Statistical differences from luciferase activity of wild type Irak-m promoter-luciferase reporter in the cells stimulated with CpG DNA (^** p<0.005) or IFNγ (^## p<0.005) are indicated. Panel B. RAW264.7 cells were transiently cotransfected with empty vector or IκB-AA and pRL-TK-luciferase plus Irak-m-promoter-luciferase (left section), NF-κB-luciferase (middle section), or CREB-luciferase (right section) reporters. Cells were stimulated with medium, CpG DNA (6 μg/ml), or IFNγ (25 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 the indicated reporter in the unstimulated cell+s) ± SD of triplicates. Statistical differences from luciferase activity of the indicated luciferase reporters in the cells transfected with empty vector and stimulated with CpG DNA (^* p<0.05) or IFNγ (^# p<0.05) are indicated. Panel C–G. RAW264.7 cells were stimulated with medium, CpG DNA (6 μg/ml), or non-CpG DNA (6 μg/ml) for 1 hr. (C) To detect NF-κB binding activity to the Irak-m promoter region, a ChIP assay was performed with anti-p50, anti-p65, or isotype control IgG Abs. DNA bound to p50 Ab, p65 Ab, or IgG was purified and used as a template for PCR with an Irak-m promoter-specific primer set that detects the region containing putative NF-κB (2) consensus site or an Irak-m-3′ end-specific primer set. Actin was used as a loading control. IP, immunoprecipitation. (D) To detect nuclear DNA binding activity of NF-κB, equal amounts of nuclear extracts (3 μg/lane) were subjected to EMSA using ³²P-labeled double-stranded ODN containing the NF-κB (2) binding sequences in the Irak-m promoter region as a probe. To detect the presence of IκBα and IκBβ, equal amounts of cytoplasmic extract (15 μg/lane) were subjected to SDS-PAGE followed by Western blot analysis. (E) Equal amounts of nuclear extracts (3 μg/lane) were subjected to EMSA using ³²P-labeled double-stranded ODN containing the wild type (Wt) or mutant (Mut) NF-κB (2) binding sequences in the Irak-m promoter region as a probe. (F) Equal amounts of nuclear extracts (3 μg/lane) were incubated with isotype control IgG or anti-p65 Ab (1 μg) for 30 min at room temperature and then subjected to EMSA using ³²P-labeled double-stranded ODN containing the NF-κB (2) binding sequences in the Irak-m promoter region as a probe. (G) Equal amounts of nuclear extracts (3 μg/lane) were incubated with an excess amount (50 X) unlabeled double-stranded ODN containing the wild type (M-Wt) or mutant (M-Mut) NF-κB (2) binding sequences in the Irak-m promoter region, universal NF-κB consensus (NF-κB), or universal AP-1 consensus (AP-1) for 30 min at room temperature and then subjected to EMSA using ³²P-labeled double-stranded ODN containing the NF-κB (2) binding sequences in the Irak-m promoter region as a probe. Panel H. RAW264.7 cells were transiently cotransfected with full length or site-directed mutants at the NF-κB (2) (−1098/-1088) or NF-κB (1) (−336/−326) sites of Irak-m-promoter-luciferase reporters and pRL-TK-luciferase and then stimulated with medium or CpG (6 μg/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 represent the mean RLU (fold induction from luciferase activity of wild type Irak-m promoter-luciferase reporter in the unstimulated cells) ± SD of triplicates. Statistical differences from luciferase activity of wild type Irak-m promoter-luciferase reporter in the cells stimulated with CpG DNA are indicated (^* p<0.05; ^** p<0.005). All experiments were repeated at least three times with similar results.