FIGURE 6.

C-terminal dsRBD, but not N-terminal helicase domain, of Dicer-2 is important for siRNA production in ovary lysate in vitro. (A) Western blotting of the ovary lysates used in the in vitro assays in Figures 6 and 7. Anti-HA antibody detects HA-tagged transgenic Dicer-2 proteins, while anti-Dicer-2 antibody detects the transgenic proteins and the endogenous wild-type Dicer-2. αTubulin and Vasa served as loading controls. (B,C) In vitro dicing assay using the fly ovary lysates. (B) Body-labeled 104-bp dsRNA with 2-nt 3′ overhang and 5′ monophosphate and (C) 5′ labeled 30-bp dsRNAs with 2-nt 3′ overhang and 5′ monophosphate were tested. The other end of the 30-bp dsRNA was blocked by two deoxynucleotides (Cenik et al. 2011). Representative gel images (left panels) and quantification of the signals (right panels). Plotted until the 60-min time points (since at 120 min, significant nonspecific degradation of the substrates were observed) are shown. Data are mean ± SD for three independent trials.