Fig. 5

cDOPEY2 serves as a scaffold to enhance the ubiquitination and degradation of CPEB4 mediated by TRIM25. A The relative mRNA levels of CPEB4 in the indicated cells as determined by qPCR. B Western blot analysis of CPEB4 stability in Eca109 cells with or without cDOPEY2 knockdown after treatment with cycloheximide (CHX, 100 µg/mL). C Western blot analysis of CPEB4 stability in control and cDOPEY2-overexpressing Eca109-CR cells treated with or without MG132 (100 µM). D TRIM25 was identified as a binding partner of cDOPEY2 by MS. E IP analysis of CPEB4 ubiquitination in ESCC cells with cDOPEY2 overexpression or knockdown. F Western blot analysis of CPEB4 in ESCC cells transfected with the indicated constructs. G FISH immunofluorescence showing the colocalization of cDOPEY2 and TRIM25. Scale bars: 20 μm. H RIP (top panel) and biotinylated probe pulldown (bottom panel) assays were performed to detect the interaction between TRIM25 and cDOPEY2. I-J. Western blot analysis of CPEB4 in ESCC cells transfected with the indicated constructs. K IP analysis of the interaction between CPEB4 and TRIM25 in 293T cells transfected with the indicated constructs. L A schematic diagram illustrating the construction of myc-tagged WT and truncated (deletion of the RNA-binding domain) TRIM25. M IP analysis of CPEB4 ubiquitination in 293T cells transfected with the indicated constructs. Data are presented as the mean ± SD. P values were determined using the unpaired Student’s t-test (A)