The immunofluorescence procedure was performed as the same as that used for above

ficient cells and mouse embryonic fibroblast were maintained in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum, penicillin G, and streptomycin. Bone marrow-derived macrophages were obtained from the bone marrow of tibia and femur by flushing with DMEM. The cells were cultured in DMEM supplemented with 20% FBS, and 30% L929 supernatant for 5 days. Knockdown of MEKK3 Oligonucleotides encoding either scrambled or MEKK3-specific small hairpin RNAs were cloned into pSUPER to generate & 2013 European Molecular Biology Organization pSUPER-scrambled or pSUPER-MEKK3 respectively. In all, 1 mg of pSUPER-scrambled or pSuper-MEKK3 was transfected into 293-I1A cells along with 0.1 mg of pBabe-puromycin by FuGENE 6. Two days after transfection, puromycin containing DMEM was added to the cells to select for puromycinresistant clones. After 10 days of puromycin selection, single clones were picked and subjected to western analysis to determine the levels of MEKK3. Clones with over 85% knockdown of MEKK3 were pooled as MEKK3 knockdown cells and used for experiments. Transfection and luciferase assay Transfection of the 293-I1A cells was performed using the FuGENE 6 transfection reagent as recommended by the manufacturer. After 24 h, the cells were stimulated with IL-1b or left untreated for 6 h before harvest. Luciferase activity was determined IRAK-M mediates TLR/IL-1R-induced NFjB activation and cytokine production H Zhou et al & 2013 European Molecular Biology Organization IRAK-M mediates TLR/IL-1R-induced NFjB activation and cytokine production H Zhou et al Bcl-2AD1 was examined by SYBR GREEN PCR Master Mix. PCR amplification was performed in triplicate, and water was used to replace cDNA in each run as a negative control. The reaction protocol included preincubation at 951C to activate PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19828299 FastStart DNA polymerase for 10 min, amplification of 40 cycles that was set for 15 s at 951C, and annealing for 60 s at 601C.First, four homology models of the IRAK-M DD were superposed onto each IRAK-2 DD segment in the Myddosome complex using the secondary structure matching algorism, as implemented in COOT. Second, these four IRAK-M DD models were assembled into the coordinates of the MyD88 Neuromedin N web DDIRAK-4 DD complex in the Myddosome complex after removal of the regions of IRAK-2 DD. Thus, the integrity and physicochemical property of the MyD88 DDIRAK-4 MM complex were maintained. The hypothetical IRAK-4 DDIRAK-M DD interface was examined by the server PISA and inspected by the program PYMOL. Co-immunoprecipitation and immunoblotting BMDMs and 293-I1A cells were cultured as indicated above. After stimulation with R848 or IL-1b, the cells were harvested and co-immunoprecipitation and immunoblotting were performed as previously described. Electrophoretic mobility shift assay BMDMs and 293-I1A cells were cultured as indicated above. After stimulation with R848 or IL-1b, the cells were harvested and nuclear extracts were prepared with the NE-PER Nuclear and Cytoplasmic Extraction Reagent. Labelling of NFkB-specific oligonucleotides was performed with -ATP by using T4 polynucleotide kinase following manufacturer’s guideline. After purification over a Sephadex G-25 column, radiolabelled oligonucleotides were incubated with nuclear extracts at room temperature for 30 min in binding buffer containing 12 mM HEPES, 4 mM TrisHCl, 60 mM KCl, 1 mM ethylenediamine tetraacetic acid, 1 mM DTT, 1 mM PMSF, 12% glycerol, 5 mg bovine serum albumin and 2 mg pol