Fig. 3

Circ-HuR interacts with CNBP protein in gastric cancer. a Coomassie bright blue staining (left panel), mass spectrometry (MS) assay, and overlapping analysis (Venn diagram, right panel) with established RBP and TF databases revealing the proteins pulled down by biotin-labeled linear or circular forms of circ-HuR from the lysates of AGS cells. b RIP and real-time qRT-PCR assays showing the relative interaction between circ-HuR and six proteins in AGS cells stably transfected with empty vector (circ-Mock), hsa_circ-23897 (circ_23897), linear circ-23897 (lin_23897), circ-HuR, or linear circ-HuR (lin-HuR), with normalization to input of cells transfected with circ-Mock. c RIP assay with primer sets (lower panel) indicating the interaction between circ-HuR and CNBP in AGS cells stably transfected with circ-Mock, circ_23897, lin_23897, circ-HuR, or lin-HuR (upper panel). d MS assay depicting the identified CNBP peptides pulled down by circ-HuR. e Dual RNA-FISH and immunofluorescence staining assay indicating the co-localization of circ-HuR (green) and CNBP (red) in AGS and MKN-45 cells, with nuclei staining with DAPI (blue). Scale bar, 5 μm. f RNA EMSA determining the interaction between endogenous CNBP protein and biotin-labeled circular probe of circ-HuR (arrowheads), with CNBP antibody incubation or competition using an excess of unlabeled circular probe of circ-HuR. g Schematic diagram revealing the domains of CNBP truncations. h In vitro binding assay showing the enriched circ-HuR levels detected by RT-PCR (lower panel) after incubation with full-length or truncations of Flag-tagged or GST-tagged recombinant CNBP protein validated by western blot (upper panel). ANOVA analyzed the difference in (b). *P < 0.01 vs. circ-Mock. Data are shown as mean ± SEM (error bars) and representative of three independent experiments in (b, c, e, f, and h)