Rip assay kit

RNA immunoprecipitation assays were performed using an RIP assay kit (Millipore Corp., Billerica, MA, USA). According to the manufacturer's protocol, the protein-RNA complex was isolated, and anti-AGO2 or IgG was added to the reaction system for immunoprecipitation. After RNA purification, the immunoprecipitated RNA was subjected to QPCR and/or PCR. IgG served as a negative control.

RNA immunoprecipitation assays were examined using a RIP assay kit (Millipore Corp, Billerica, MA, USA). According to the manufacturer's protocol, the protein‐RNA complex was isolated, and anti‐AGO2 or IgG was added to the reaction system for immunoprecipitation. After RNA purification, the immunoprecipitated RNA was subjected to QPCR and/or PCR. IgG served as a negative control.

The interaction among miR‐140‐5p, PGM5‐AS1, and FBN1 was identified with a RNA immunoprecipitation (RIP) assay kit (Millipore Inc., Bedford, MA, USA). Following treatment with lysis buffer (P0013B; Beyotime Biotechnology Co., Shanghai, China), the cells were subjected to 10‐min centrifugation at 35 068 g and 4 °C to harvest the supernatant. A large portion of cell extract was taken as the input, and the remainder was cultured with the antibody for coprecipitation as follows: After being resuspended in 100 μL RIP Wash Buffer, 50 μL magnetic beads were cultured with 5 μg antibody. The complex of bead and antibody was then resuspended using 900 μL RIP Wash Buffer, followed by overnight culture in 100 μL cell extraction at 4 °C. The beads–protein complex was obtained by placing the samples on the magnetic base. The antibodies used for the RIP assay were argonaute 2 (Ago2, ab32381, 1 : 50; Abcam Inc.), with IgG (1 : 100, ab109489; Abcam Inc.) used as the NC.

According to the manufacturer’s instructions, RIP assay kit (Millipore, USA) was used for RIP assay. Briefly, HEK-293 cell suspension was prepared in RIP buffer. Anti-FOXO1 antibody (Cell Signaling Technology, 5 μg) was incubated with the cell suspension at 4 °C overnight. Then, the precipitated RNA was purified and analyzed by qRT-PCR. Isotype-matched IgG (5 μg) was used as a negative control.