An RIP assay was performed using a Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) [6 (link)]. After the transfected MCF7 and MDA-MB-453 cells were harvested, the cells were lysed using RIP lysis buffer (Solarbio, Beijing, China). Next, we added magnetic beads (Invitrogen) to RIP lysis buffer (Solarbio) and then conjugated the beads with anti-Ago2 (Abcam) or anti-IgG (Abcam) (overnight, 4°C). After digestion with proteinase K (Absin, Shanghai, China) the immunoprecipitated RNA was acquired and then quantified by RT-PCR.
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