Pull-down Assay for miR-181a-5p Regulation

Pull-down assays were performed as described previously [27 (link)]. Briefly, S1-CRNDE and S1-CRNDE mutant were generated, and cotransfected with or without miR-181a-5p inhibitor. After 48 h of transfection, cells were harvested and washed by PBS for two times, then crosslinked in 0.37% formaldehyde, incubated in ice-cold lysis buffer (150 mM NaCl, 10 mM Hepes, 3 mM MgCl₂, 10% glyceral, 1% NP-40, 2 mM DTT, 1 mM PMSF, 1 × protenase inhibitor (Sigma), 10 ul RNase inhibitor (promega)). The cell lysate was precleaned in agarose beads (Santa Cruz Biotechnology) at 4 °C for 1 h, then incubated and rotated in streptavidin beads (Thermo Fisher Scientific, San Jose, CA, USA) at 4 °C for 3 h. The streptavidin beads were collected, washed in elution buffer (50 mM Hepes, 5 mM EDTA, 100 mM NaCl, 1% SDS, 10 mM DTT). The beads were heated at 70 °C for 45 min. miR-181a-5p expression was examined by qRT-PCR.