Anti immunoglobulin g igg

MDA-MB-231 cells were transfected with negative control or miR-5088-5p mimic. After 48 h, cells were washed by ice-cold PBS for 10 min to washing. Ago2-IP assay was performed using the RiboCluster Profiler/RIP (Ribonucleoprotein IP)-Assay Kit for miRNA (MBL International, Nagoya, Japan). The samples were lysed with lysis buffer containing a protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA) and centrifugation at 12,000 × g at 4°C for 5 min. The precleared lysates and beads were prepared and mixed with anti-human Ago2 (Sigma-Aldrich, St. Louis, MO, USA) and anti-immunoglobulin G (IgG) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as control overnight at 4°C using a rotator. Ago2-IP beads were washed with ice-cold wash buffer 3 times. After RNA extraction by the manufacturer’s instructions, a real-time PCR assay was performed using miR-5088-5p primer.