RNA immunoprecipitation (RIP) assay
The RIP assay was performed using a Magna RIP RNA Binding Protein
Immunoprecipitation Kit (Bersinbio, China) according to the
manufacturer’s protocol. 2×107 MG63 or U2OS cells were
lysed in complete RIP lysis buffer and the cell lysates were divided
into two equal parts and incubated with either 5 μg human
anti-Argonaute2 (AGO2) antibody (Millipore, USA) with rotation at 4°C
overnight. Magnetic beads were added to the cell lysates and incubation
was continued at 4°C for 1h. The samples were then incubated with
Proteinase K at 55°C for 1h. The enriched RNA was obtained using RNA
Extraction Reagent (Solarbio, CHN). The purified RNA was used to detect
the expression levels of the genes of interest by qRT-PCR.