FIGURE 3.

Association of Argonaute N-PAZ variants with small RNAs. A, FLAG-tagged DmAgo1 variants (WT, PAZ6, and ΔN-PAZ) were expressed in S2 cells and immunoprecipitated. Co-purification of bantam miRNA was assessed by Northern blot analysis. A mock transfection served as a negative control. B, RNA that co-purified with DmAgo1 variants was radiolabeled with [γ-³²P]ATP and T4 polynucleotide kinase and resolved on denaturing PAGE and exposed to phosphoimaging screen. C, detection of small RNA species associated with DmAgo1 in co-IP by RNase protection. RNA associated with DmAgo1 wild type or variants was extracted, hybridized with 43-nt radioactive body-labeled RNA containing a perfect bantam binding site, and digested with RNase specific for single-stranded RNA. The digestion products were then resolved by PAGE and exposed to a phosphorimaging screen. As a control, the probe was digested in the presence of 1 pmol of bantam miRNA (23 nt) or without any RNA. CTR, control.