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. Author manuscript; available in PMC: 2012 Jan 1.
Published in final edited form as: Nat Med. 2010 Dec 5;17(1):64–70. doi: 10.1038/nm.2266

Figure 4.

Figure 4

Validation of downstream target genes for miR-124. (a) Alignment of three predicted miR-124 binding sites to C/EBP-α 3′ UTR is shown for different species (Mus musculus, Homo sapiens, Pan troglodytes, Macaca mulatta, Rattus norvegicus and Oryctolagus cuniculus). (b) Western blot analysis of C/EBP-α expression in BMDMs transfected with miR-124 or control miRNA (c) Flow cytometry analysis of the expression levels of C/EBP-α and CD45, or PU.1 and CD45 in BMDMs transfected with miR-124 or control miRNA. Percentages of CD45hi C/EBP-α+ and CD45hiPU.1+ cells are shown in upper right quadrants. Populations of miR-124–transfected CD45low cells were negative for C/EBP-α and PU.1 expression, as shown in double staining for cell-surface CD45 and intracellular C/EBP-α or PU.1 (lower left quadrants). Staining for CD45 (x axes) and either C/EBP-α, PU.1 or corresponding isotype controls (y axes) are shown. (d) The mean ± s.e.m. of percentages of CD45hi C/EBP-α+ and CD45hiPU.1+ cells from four independent experiments. **P < 0.01. (e) Luciferase activity in NIE115 cells transfected with reporter constructs containing either intact or mutated C/EBP-α 3′ UTR. The NIE115 cell line was co-transfected with the indicated constructs and either miR-124 or control miRNA, and normalized levels of luciferase activity are shown.