Protein g sepharose 4 fast flow suspension

Transfected cells were lysed in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate) containing RNase inhibitors and protease inhibitors (Thermo Scientific, Waltham, MA, USA). The extracted protein was incubated with relevant antibodies (anti-ELAVL1 and IgG as control) (Abcam, USA) overnight at 4 °C and then pulled down with protein G Sepharose 4 Fast Flow suspension (GE Amersham, Little Chalfont, UK). The beads were digested with proteinase K (Sangon, Shanghai, China) for 1 h followed by RNA purification with Trizol reagent (Invitrogen, Missouri, USA). qRT-PCR was performed to examine the RNA yield. The primers were listed in the qRT-PCR section.

An EZMagna RIP kit (Millipore, USA) was used to perform RIP analysis following the manufacturer’s protocol. Briefly, 10 µL (10%) cell lysate supernatant was used as input. The rest of cell lysates were incubated with specific antibodies (anti-Ago2, 1:1 000 dilution and IgG as control) (Abcam, MA, USA) overnight at 4 ℃, followed by pulling down with protein G Sepharose 4 Fast Flow suspension (GE Amersham, Little Chalfont, UK). The beads were washed with lysis buffer and digested with proteinase K (Sangon, Shanghai, China) for 1 h. The elutes were used for RNA purification with Trizol reagent (Invitrogen, Missouri, USA), RT-qPCR was performed to analyse the levels of specific RNA transcripts.