Anti ago1

Co-immunoprecipitation (Co-IP) of miRNP with anti-Ago1 (Abcam, Cambridge, MA, USA) or IgG (Sigma) was performed as previously described (Tan et al, 2009 (link)). RNA co-immunoprecipitated with anti-Ago1 or IgG antibodies was extracted using TRIzol LS (Invitrogen) as described in a previous publication (Cheng et al, 2008 (link)). Total RNA was subjected to DNA digestion as described above.

Kc167 cells were stably transfected with a plasmid containing FLAG-HA tagged AGO2 genomic fragment (Czech et al. 2008 (link)). Immunoprecipitation was carried out using anti-FLAG (Wako) or anti-AGO1 (AbCam) antibody in RIPA buffer as described previously (Okamura et al. 2013 (link)). For detection of miR-10404/miR-ITS1-5p, a DNA probe (Fig. 3A) or an LNA probe (Fig. 3B,C,D) was used. Small RNA enrichment was performed for the top two panels of Figure 3C and the top three panel of Figure 3D using mirVana miRNA isolation kit (Ambion). Oligo probe sequences are listed in Supplemental Table S5. Northern blotting and preparation of pasha mutant samples were described previously (Okamura et al. 2008 (link); Martin et al. 2009 (link)).

RNA immunoprecipitation (RIP) assay was performed with anti-Ago1 (Abcam, Cambridge, MA, USA) or IgG (Sigma-Aldrich). SMMC7721 cells at 48 h after transfection with miR-506 or miR-NC were lysed in complete RIP lysis buffer, and then 100 µL whole-cell lysate of SMMC7721 cells was incubated with RIP buffer containing magnetic beads conjugated with human anti-Ago1 or negative control IgG. Samples were incubated with Proteinase K with shaking to digest the protein, and RNA coimmunoprecipitated with anti-Ago1 or IgG antibodies was isolated using TRIzol (Invitrogen) and then subjected to qRT-PCR analysis.

Magna RIP RNA-Binding Protein Immunoprecipitation Kit (cat. no. 17–700; Sigma-Aldrich; Merck KGaA) was used for the RIP assay. Briefly, A549 or NCIH1385 cells (1×10⁷) were lysed in RIP lysis buffer (Beyotime Institute of Biotechnology) on ice for 30 min, and then supernatant was incubated with 30 µl of Protein-A/G agarose beads (Roche Diagnostics) supplemented with 2 µg anti-Ago1 (product code ab279392; 1:300) or anti-IgG (product code ab238004; 1:300; both from Abcam) at 4°C overnight. The beads were washed 5 times with RIP washing buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, and 0.5% NP-40), and centrifuged at 2,000 × g for 1 min at 4°C. The bounded proteins were boiled with 1X SDS loading buffer and analyzed by western blotting, and immunoprecipitated RNAs were analyzed by RT-qPCR.