Research Paper Volume 11, Issue 22 pp 10220—10241

Circular RNA CircCCNB1 sponges micro RNA-449a to inhibit cellular senescence by targeting CCNE2

Figure 8. Identification and characterization of CircCCNB1-binding proteins (CBPs). (A) Schematic illustration of the modified RNA pulldown. Briefly, an MS2-tagged RNA was introduced into a CircRNA to express this specific tag(CircRNA-MS2) in cells; A Flag-tagged MS2-capturing protein(flag-MS2-CP) was constructed to specifically interact with the MS2 tag to capture CircRNA-MS2; Immunoprecipitation of the flag-MS2-CP-CircRNA-MS2 complex was performed using the flag antibody to identify proteins or miRNA molecules that might interact with CircRNA. (B) qRT-PCR detected the relative enrichment of circRNA-MS2 in HEK293T after transfection with the indicated plasmids, ***P<0.001. (C) Immunoprecipitation (IP) and immunoblot identification of CBPs.