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. 2012 Aug 16;8(8):e1002872. doi: 10.1371/journal.ppat.1002872

Figure 6. VP1 interferes with the effector phase of the RNAi pathway.

Figure 6

(A) Mobility shift assays for binding of viral RNAi suppressor proteins to siRNAs. Radiolabeled siRNAs were incubated in buffer (lane 1) or with decreasing amounts of recombinant MBP-VP1ΔN284 (lanes 2–5), MBP (lanes 6–9), and MBP-NS3 (lane 10–13). Ten-fold dilutions were used, starting at 2 µM for MBP-VP1ΔN284 (lane 2) and 2.6 µM for MBP (lane 6). MBP-NS3 was tested in two-fold dilutions (highest concentration of 8 µM, lane 10). RNA mobility shifts were analyzed on an 8% native polyacrylamide gel. (B) RISC Slicer assay in Drosophila embryo lysate. Lysates were incubated with non-targeting control siRNA (Ctrl, lane 1) or with Fluc siRNA (lanes 2–4) in the absence (lane 2) or presence of recombinant MBP-VP1ΔN284 (lane 3) or MBP (lane 4). RISC cleavage products were analyzed on an 8% denaturing polyacrylamide gel. Slicer assay is representative for two independent experiments.