Fig. 4

CircFAM13B interacts with IGF2BP1 protein via the KH3–4 domain. A RNA–protein pulldown experiment and silver staining assay were conducted to investigate the possible proteins, which could bind circFAM13B. B Mass spectrometry assay indicated that IGF2BP1 was pulled down by the circFAM13B probe. C GO enrichment analysis showed that the proteins pulled down by circFAM13B probe were enriched in the mRNA stability regulation pathway. D The results of StarBase database predictions and mass spectrometry analysis were intersected, and IGF2BP1 binding to both circFAM13B and PKM2 was found. E The binding site of circFAM13B and IGF2BP1 was predicted using the catPAPID algorithm. F RNA–protein pulldown and Western blot assays were performed to confirm that circFAM13B could bind to IGF2BP1. G RIP assays in circFAM13B overexpression and relative control T24 cells were conducted to validate the binding of IGF2BP1 and circFAM13B (**P < 0.01, ***P < 0.001, Student’s t-test). H Full length or truncations of flag-tagged recombinant IGF2BP1 protein vectors were designed and constructed. I The vectors containing full length or truncations of flag-tagged recombinant IGF2BP1 were transfected into T24 cells successfully. J RIP assays and qRT-PCR were carried out by using flag antibodies to determine the region of IGF2BP1 that could bind to circFAM13B. K Nuclear-cytoplasmic fractionation and qRT-PCR were performed to validate whether circFAM13B and IGF2BP1 are located in the nucleus or cytoplasm. L IF-FISH assays were carried out to indicate the co-localisation of circFAM13B and IGF2BP1 in T24 cells. M The expression of IGF2BP1 in BCa tissues and adjacent normal tissues were detected by qRT-PCR (**P < 0.01, Student’s t-test). Pearson’s correlation analysis was conducted to validate the correlation of circFAM13B and IGF2BP1. Data are expressed as mean ± SD, n = 3