Fig. 3

cDOPEY2 physically interacts with CPEB4 protein. A Identification of cDOPEY2-protein complexes pulled down by a probe targeting the splicing junction of cDOPEY2 with a protein isolated from ECA109 cells. The arrows indicate the band corresponding to the top-hit protein stained with Coomassie Blue. B The top-hit protein enriched with cDOPEY2 as determined by MS analysis. C Bubble plot of the top 10 BP terms identified by the GO enrichment analysis of proteins regulated by cDOPEY2 in ECA109 cells. D Volcano plot illustrating the dysregulated proteins of interest (fold change > 4 and P < 0.01) in the cDOPEY2-overexpressing group compared with controls in ECA109-CR cells. E Venn diagram illustrating the overlapping proteins that were both enriched and regulated by cDOPEY2 as determined by MS analysis. F A schematic diagram showing the CPEB4 binding motif (UUUUA) of cDOPEY2 at 317-322 nt. G A biotinylated probe pulldown assay was performed to detect the interaction between CPEB4 and cDOPEY2 in ECA109 cells. H RIP assays were performed to detect the interaction between CPEB4 and cDOPEY2 in ECA109 cells. Bottom, IP efficiency of the CPEB4 antibody. Top, the abundance of CPEB4-associated cDOPEY2 was analyzed by qPCR and normalized to the input control. An IgG antibody was used as a negative control. I FISH immunofluorescence showing the colocalization of cDOPEY2 and CPEB4. Scale bars: 20 μm. J A schematic diagram illustrating the construction of myc-tagged CEPB4 with different truncations. K Left panel: western blotting was performed to assess the IP efficiency of the myc antibody in ECA109 cells with plasmids encoding myc-tagged WT or truncated CPEB4. Right panel: relative enrichment of cDOPEY2 in ECA109 cells with plasmids encoding myc-tagged WT or truncated CPEB4 as determined by qPCR. L A biotinylated probe pulldown assay was performed to detect the interaction between CPEB4 and mutant cDOPEY2 in 293T cells