RNA-Protein Interaction Identification

RNAs were biotin-labeled during in vitro transcription using Biotin RNA Labeling Mix (Roche) and T7 polymerase (New England Biolabs). P-18 primary cells cultured in androgen-depleted medium were lysed in modified Binding buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS and 1% protease inhibitor cocktails). Cell lysates were incubated with biotin-labelled RNAs and streptavidin beads at 4 °C for 12 h. The beads were washed in wash buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 0.05% Nonidet P-40 (NP-40); 1 mM MgCl2) at 4 °C six times. The samples were subjected to western blot analyses as described previously [34 (link)]. Briefly, protein samples were denatured and subjected to SDS-polyacrylamide gel electrophoresis (SDS/PAGE), and were transferred to nitrocellulose membranes (Bio-Rad). The membranes were immunoblotted with specific primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and visualized by SuperSignal West Pico Stable Peroxide Solution (Thermo Scientific). The antibodies are shown in Table S1.