RNA-Protein Interaction Purification

As previously described 43 (link), the RNA affinity purification was performed. The 5'biotin-labeled PKM EI9 RNAs were synthesized by Genscript company (Nanjing, China) according to Chen et al.'s research 29 (link). 1 nmol biotin-labeled RNAs was bound with 50 μl Streptavidin-Agarose beads (Sigma, GE17-5113-01) to prepare RNA-immobilized beads. Furthermore, cells grown at 70-80% confluency were transfected with the indicated plasmid for 48h. Cells were lysed in IP lysis buffer (beyotime, P0013) adding 1× phosphatase inhibitor cocktail (Sigma-Aldrich & Roche). Nuclear pellets were collected with Nuclear and Cytoplasmic Protein Extraction Kit, and lysed by sonication. Nuclear lysates were incubated with RNA-immobilized beads at 30 °C for 30 min while rotating. After protein and RNA binding, proteins were eluded with using SDS lysis buffer (beyotime, P0013G). The samples were detected by Western blotting.