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. 2019 Sep 16;9:13393. doi: 10.1038/s41598-019-49706-9

Figure 7.

Figure 7

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).