Multitargeting of the TLR4 signaling pathway by miR-146b. 3′UTR luciferase constructs were cotransfected in 293T cells with miR-146b mimic or a negative control mimic (ctrl). MyD88 (A), IRAK-1 (E), TRAF6 (I), and IL-6 (M). Results are expressed as the ratio between renilla and firefly luciferase activities (mean percent variation ± SEM; n = 3). Cell extracts from pRRL-ctrl–transduced (CT) or pRRL-146b–transduced (146b) THP-1 cells (white histograms); miRzip-ctrl–transduced or miRzip-146b–transduced THP-1 cells (black histograms) were subjected to RIP assay by using anti-Ago2 or IgG control Abs. Levels of MyD88 (B), IRAK-1 (F), TRAF6 (J), and IL-6 (N) mRNAs were assayed in triplicate by qPCR in RIP (IP AGO2) and leftover samples and expressed as normalized fold enrichment. (mean percent variation ± SEM; n = 3). Protein levels were analyzed by Western blot and normalized on tubulin, used as loading controls. MyD88 (C and D), IRAK-1 (G and H), and TRAF6 (K and L). One experiment representative of four is shown.