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. 2004 Jul 15;18(14):1655–1666. doi: 10.1101/gad.1210204

Figure 5.

Figure 5.

AGO1 is required for efficient miRNA-initiated RNA cleavage. (A) When AGO2 is suppressed by introducing specific dsRNA in S2 cells expressing EGFP, the ability of the cells to silence EGFP by RNAi is severely reduced. In contrast, when AGO1 is suppressed, the EGFP silencing effect is unaffected, indicating that AGO1 is not essential for the RNAi pathway in S2 cells. (B) Lysates were prepared from 14- to 16-h yw, AGO2414 mutant, and AGO1k08121 mutant embryos. Western blots were performed using the antibodies against AGO2 (upper), AGO1 (middle), and ribosomal protein P0 (lower) as a loading control. (C) In vitro RNAi assays with lysates prepared from 14- to 16-h yw, AGO2414, and AGO1k08121 embryos. The RNA target was the same as that used in Figure 4B. The amount of specifically cleaved bands was quantified and normalized to that of bands in wild-type embryos. (D) AGO1 is not required for the production of the siRNA duplex. Uniformly 32P-labeled dsRNA corresponding to the ftz gene, incubated in extracts from yw and AGO1k08121 embryos. RNA products of the reaction were analyzed on a polyacrylamide gel. Incubation of lysates with labeled dsRNA generates RNA fragments ∼22 nt long. [WT(yw)] Yellow-white wild type; (AGO1k08121) AGO1k08121 embryos. (E) RISC formation is not impaired in AGO1k08121 embryo lysates.