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. 2013 Jun 24;110(28):11499–11504. doi: 10.1073/pnas.1219852110

Fig. 4.

Fig. 4.

TLR4 is a direct target of miR-146b. (A and E) Luciferase constructs with the entire 3′UTR of TLR4 (luc-TLR4) or the corresponding construct mutated in the miR-146b seed region (luc-mut-TLR4) or TLR2 (luc-TLR2) were cotransfected in 293T cells with miR-146b mimic or a negative control mimic (ctrl). Results are expressed as the ratio between renilla and firefly luciferase activities (mean percent variation ± SEM; n = 3). (B and F) Cell extracts from THP-1 cells transduced with pRRL-ctrl (CT; closed columns) or pRRL-146b (146b; closed columns) or transduced with miRzip-ctrl (CT; open columns) or miRzip-146b (146b; closed columns) were subjected to RIP assay by using anti-Ago2 or IgG control Abs, and levels of TLR4 and TLR2 transcripts (B and F, respectively) were assayed in triplicate by qPCR in RIP (IP AGO2) and leftover samples. Results are expressed as normalized fold enrichment (mean percent variation ± SEM; n = 3). (C, D, G, and H) Protein levels were measured by flow cytometry on THP-1 cells transduced with pRRL-ctrl (gray histogram) or pRRL-146b (black histogram) (C and G, TLR4 and TRL2, respectively) or with miRzip-ctrl (gray) or miRzip-146b (black; D and H, TLR4 and TRL2, respectively). The isotype control staining is shown by the white histogram. One experiment representative of four performed with similar results is shown.