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. Author manuscript; available in PMC: 2013 Dec 8.
Published in final edited form as: Cell Rep. 2013 Oct 24;5(3):10.1016/j.celrep.2013.09.029. doi: 10.1016/j.celrep.2013.09.029

Figure 4. Trimming is dispensable for target silencing in vitro and in cultured cells.

Figure 4

(A) FLAG-Ago2 loaded with ac-pre-miR-451 was incubated with PARN-overexpressing HeLa S3 lysate (+Trimming) or buffer alone (−Trimming) at 37 °C for 3 h. Trimming was confirmed by Northern blotting

(B) Target cleavage was comparable with or without trimming. The graph shows means and standard deviations from three independent cleavage reactions.

(C) Schematic representation of the Renilla luciferase reporters used in Figure 4E for cultured cells.

(D) Inhibition of trimming by three phosphorothioate linkages in HeLa S3 cells. Pre-miR-451 or pre-miR-451 3×PS was transfected and Ago2-bound RNA was immunopurified and analyzed by Northern blotting.

(E) Renilla luciferase reporter assay for 1× perfect target site, 2× centrally mismatched target sites or CAB39 3′ UTR containing an endogenous miR-451 target site by pre-miR-451 with or without 3×PS. Firefly luciferase served as a control. The Rluc/Fluc luminescence was normalized to the value of mock transfection. The mean values ± standard deviations from three independent experiments are shown. Trimming was dispensable for target silencing in cell culture.