RIP Assay for miR-375 Targeting

RIP assay was performed using the Protein A/G Agarose Resin (YEASEN, China) following the manufacturer’s protocol. For RIP assay, SLC7A11 3′UTR sequences containing miR-375 binding sites or mutated binding sites were inserted into pcDNA3.1 (+) plasmid, named as SLC7A11 3′UTR (WT) or SLC7A11 3′UTR (MUT), respectively. SGC-7901 and BGC-823 cells were transfected with SLC7A11 3′UTR (WT) and SLC7A11 3′UTR (MUT). After 24 h, cells were extracted and lysed by NP-40 lysis buffer (Beyotime, China). After that, 100 μL lysate was incubated with NP-40 buffer containing Protein A/G Agarose Resin bound with human anti-Ago2 antibody (Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. Agarose resin was isolated by centrifugation and digested with protease K (YEASEN) to isolate Ago2-RNA complex from the beads. qRT-PCR was performed to detect miR-375 level [44 (link)].