Effect of miR-146a on NF-κB translocation. HeLa cells were first treated with miR-146a mimics, AMO-146a, and scramble RNAs, respectively. The cells were treated with LPS at 18 h and harvested 24 h after the transfection. (A–D) Total, cytoplasmic, and nuclear portions of the p65 subunit of NF-κB were analyzed by western blotting. Total and cytoplasmic proteins were normalized using β-actin, whereas histidine H3 was used to normalize nuclear protein. (E,F) Detection of NF-κB activation with luciferase reporter assay. pNF-κB-Luc (Stratagene) is a plasmid in which Firefly luciferase (Luc) expression is modulated by an NF-κB response element. NF-κB activation can be measured by the level of Luc expression. HeLa cells were transfected with pNF-κB-Luc, pRL-TK, and various RNAs. The expression of Luc and RLuc in these cells was detected with dual luciferase reagents at 24 h posttransfection. (G,H) The mRNA and protein of IL-6 and TNF-α were detected with RT-qPCR and western blotting, respectively. Error bars represent SD, n = 3.
