nuclear extraction transam kit  (Active Motif)

Journal: Scientific Reports

Article Title: The reduction of miR146b-5p in monocytes and T cells could contribute to the immunopathogenesis of hepatitis C virus infection

doi: 10.1038/s41598-019-49706-9

Figure Lengend Snippet: NF-kB transcription assay and reporter assay. THP-1 cells were electroporated with 300 nM of miR-146b mimic or negative control (Ambion) and treated with TNF-α (20 ng/ml) or diluent control for 6 hours ( A ). Nuclear extracts from the cells were prepared using a TransAM Nuclear Extract Kit (Active Motif). The transcription activity of NF-kB was measured using an NF-kB p65 transcription factor assay kit (Active Motif). Bars represent mean absorbance of three determinants ± SE. *p < 0.05 ( B ). THP-1, Jurkat and PLC/PRF/5 cells were electroporated with pEZX-MT06 target reporter vectors containing 3′ UTR of NFKB1, MAP3K7 and TRAF6 (GeneCopoeia) together with miR-146b mimic or negative control (Ambion) ( C ). After 24 hours, luciferase assay was performed using Luc-Pair Luciferase Assay Kit 2.0 (GeneCopoeia). Luminescence of the firefly luciferase was normalized using the Renilla luciferase. Relative luminescence of a reporter vector co-transfected with miR-146b mimic was calculated as a ratio for negative control. Bars represent mean relative luminescence of five determinants ± SE. *p < 0.05 ( D ).

Article Snippet: The nuclear extract from the cells was prepared using a TransAM Nuclear Extract Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Transcription Assay, Reporter Assay, Negative Control, Control, Activity Assay, Transcription Factor Assay, Luciferase, Plasmid Preparation, Transfection