As compared to Vif-CBFb140-EloB/C and Cul5, the mixture had an earlier elution peak

at room temperature before use. TRIzol reagent and SuperScript VILO cDNA Synthesis Kit were from Invitrogen, Life Technologies and co-precipitant from Bioline. TURBO-DNase was from Ambion, LightCycler 480 SYBR Green I Master from Roche; other reagents for real-time RT-PCR were from Invitrogen, Life Technologies. Rabbit anti-human IkBa, p44/42 MAPK, phospho-p44/42 MAPK, phospho-MEK1/2 was treated with Turbo DNase and reverse transcribed using SuperScript VILO cDNA synthesis kit according to manufacturer’s instructions. Negative controls were prepared by performing reverse transcription reactions in the absence of Superscript Enzyme Mix and cDNA. PCR amplification for IL-6, IL-8, IL-10, TNF-a, TF, NF-kB1 and b-actin was performed with LightCycler 480 SYBR Green I Master Mix as described. Assays performed in duplicate containing 5 ml 2x SYBR Green I Master Mix, 4 ml template cDNA or negative control, 1 ml 2.5 mM forward and reverse combined primers. Reactions were amplified and quantified using the LightCycler 480 system with standard cycle conditions, and analyzed using the appropriate software. Relative quantities of mRNA in duplicate samples were S100A12 Blunts Monocyte 6145492 Cytokine Induction by SAA calculated by the comparative cycle threshold method and normalized against human b-actin mRNA as endogenous control. In addition to b-actin, real-time RT-PCR analysis of cytokine suppression by S100A12 and stability of IL-6 mRNA were normalized to HPRT as housekeeping gene and results were no different to those obtained when normalized against b-actin. To determine whether S100A12 suppression of cytokine levels was due to mRNA stability, the half-life of cytokine mRNA was measured by culturing THP-1 cells with S100A12, SAA or both for 4 h at 37uC in 5% CO2 in air. Actinomycin D was subsequently added to block transcription, and cells immediately returned to 37uC. Cells were harvested immediately or following 30, 60, 90, 120 and 180 min. Levels of IL-6 and TNFa mRNA were determined as described above. or goat anti-mouse IgG for 1 h at RT, followed by 365-min washes, and reactivity visualized by Western Lightning-enhance chemiluminescence substrate. Immunofluorescence for NF-kB p65 nuclear translocation in THP-1 cells treated with SAA 6 S100A12 was performed as described. Para-nitrophenyl Phosphate Phosphatase Assay The general phosphorylation activity of SAA 6 S100A12treated THP-1 cells was measured by assessing the total phosphatase activity using pNPP as a substrate. Stimulated cells were collected, washed once with PBS, then lysed with 250 ml reaction mixture containing 1.5 mM EDTA, 37.5 mM Na acetate, 0.15% w/v Triton X-100, 3% w/v glycerol and 5 mM DTT. For kinetic reactions, 100 ml pNPP was mixed with reaction mixture. Samples were incubated at 37uC for 30 min, then quenched with 50 ml 3 M Tris. Release 7751958 of para-nitrophenyl was determined spectrophotometrically by measuring A405 nm, and absorbance calculated as a ratio of enzyme activity relative to control. Cytokine Measurement Culture supernates from stimulated PBMC and THP-1 cells were assayed in duplicate for IL-6, IL-8, TNF-a and IL-1b levels using cytokine-specific DuoSet ELISA kits according to manufacturer’s instructions. Intracellular IL-8 levels in SAA 6 S100A12-treated THP-1 cells were determined by flow cytometry. Stimulated THP-1 cells were transferred to FACS TG100 115 web polystyrene tubes; Franklin Lakes, NJ), washed with cold PBS containing 0.5% BSA and 0.1% sodium azide, pre-fixed with