RNA Binding Protein Immunoprecipitation (RIP) Assay

CNE-2 cells were collected and washed with precooled phosphate buffer saline (PBS). Then, cells were lysed with RIP lysis buffer (P0013B, Beyotime Biotechnology Co., Shanghai, China) and ice-bathed for 5 min. RIP kit (Merck Millipore, Billerica, MA, USA) was employed to detect the binding SMAD5-AS1 to Argonaute2 (AGO2) protein. Magnetic beads in lysis buffer were incubated with 5 μg rabbit anti-human AGO2 antibody (ab186733, 1:50, Abcam, Shanghai, China) for 6 h at 4°C. After incubation, 100 μL cell lysate was added to the magnetic beads-antibody complex and incubated overnight at 4°C. Samples were placed on magnetic pedestals to collect magnetic beads-protein complexes. The samples were detached with proteinase K buffer to collect RNA, followed by RT-qPCR. The percentage of SMAD5-AS1 binding to AGO2 protein (%) was calculated with untreated cell supernatant as reference. Rabbit anti-human IgG (ab109489, 1:100, Abcam, Shanghai, China) was used as NC.