Anti ago1

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.

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.