Fig. 7
CircRYK interacts with HuR and promotes its cytoplasmic transport. (A) The Venn diagram illustrates the potential RNA-binding proteins (RBPs) that circRYK may interact with. (B) Silver staining of the circRYK pulldown. (C) HuR was confirmed to be enriched in the circRYK probe by Western blot. (D) The RIP test findings indicate that the HuR protein formed a precipitate with circRYK in GBM cell lysates. (E) The predicted interaction sites between circRYK and HuR. (F) The diagram illustrates the truncated portion of the circRYK overexpression plasmid. RNA pull-down was carried out using circRYK-specific probes after GBM cells were transfected with wild-type or truncated circRYK overexpression plasmids. (G) The levels of circRYK were determined using qRT‒PCR after treatment of U87 cell lysates with either full-length or truncated versions of Flag-labelled recombinant HuR protein. (H) RNA pull-down studies employed circRYK-specific probes with full-length or truncated versions of the Flag-tagged recombinant HuR proteins. (I) Immunofluorescence was employed to verify the expression of HuR in U87 cells after the knockdown or overexpression of circRYK. (J) Western blotting was employed to determine HuR expression levels in whole lysates or subcellular fractions. The shCONT, shcircRYK, vector, and circRYK overexpression plasmids were transfected into GBM cells. Scale bar, 100 μm. Each experiment was performed three times, and the results are displayed as the mean ± SD (****P < 0.0001)