Fig. 6

CircSTX6 interacts with the PABPC1 protein in BCa. A Silver staining depicted the proteins that interact with circSTX6. Biotin-labeled sense or antisense circSTX6 probes were used for RNA protein pull-down against EJ cells lysates. B Venn diagram demonstrated the overlapping of the interacting RBPs of circSTX6 determined by Mass spectrometry and predicted by RBPmap and RBPDB databases. The 239 proteins with molecular masses of 55–100 kDa that were only pulled down by sense probe were screened. C Mass spectrometry assay depicted the PABPC1 peptides pulled down by circSTX6 sense probes. D Western blot assay showed the PABPC1 protein pulled down by biotin-labeled circSTX6 antisense or sense probes from the lysates of BCa cells. E RIP assays showed the association of PABPC1 and circSTX6 (upper). The precipitate was subjected to western blot with the antibodies against PABPC1 (lower). IgG was used as the negative control. F The subcellular location of PABPC1 and circSTX6 in BCa cells was evaluated by IF and FISH assay. Nuclei were stained with DAPI. Scale bar, 10 μm. G The schematic structures revealed the full-length and truncations plasmids for PABPC1. H Full-length or truncations of Flag-tagged recombinant PABPC1 proteins in EJ cells were validated by western blot analysis. I Relative enrichment of circSTX6 levels was detected by RIP and qRT-PCR assays in EJ cells transfected with Flag-tag plasmids of PABPC1 full-length or truncations. J Full-length or truncations of Flag-tagged recombinant PABPC1 proteins in UMUC3 cells were validated by western blot analysis. K Relative enrichment of circSTX6 levels was detected by RIP and qRT-PCR assays in UMUC3 cells transfected with Flag-tag plasmids of PABPC1 full-length or truncations. The data are presented as the means ± S.D. of at least three independent experiments. *P < 0.05. P values are calculated by Student’s t test in E, I and K