Validation of RBPs in an mRNA pull-down assay. A) Flowchart of the RNA pull-down assay. B) Establishment of the RNA pull-down assay using the known RBP HLP1 as the positive control. Recombinant MBP-His-HLP1 was detected by the anti-His antibody (Lane 3). MBP-His and beads without RNAs were used as negative controls (Lanes 2 and 4). C) Full-length ATSDH and ESM1 bind RNAs in vitro. MBP-His-ATSDH/ESM1 was recognized by the anti-His antibody. D to H) Interaction of 5 non-ploy(A) RBPs (Lane 2, left panel) and their enzymatic domains (Lane 2, right panel) with RNAs. I) Interaction between non-poly(A) RBPs and polyadenylated non-poly(A) RNAs. All recombinant proteins detected by the immunoblot are indicated by *.