Fig. 4: CDC25A is phosphorylated by DYRK2.

A Purified CDC25A protein was incubated with GST-DYRK2-WT, GST-DYRK2-KD, or GST alone. Phosphorylation was determined by autoradiography, while CDC25A and DYRK2 were detected by WB with antibodies to GST and CDC25A, respectively. B IVK assay performed with purified DYRK2 and CDC25A proteins. CDC25A phosphorylation was determined by WB with a phospho-S/T-Pro antibody, while total protein levels of DYRK2 and CDC25A were assessed by WB. C TOP: Schematic CDC25A representation showing different functional regions and the phosphorylated amino acids detected (in yellow). BOTTOM: Extracts from transfected HEK-293T cells with plasmids to express Flag-CDC25A-WT or the indicated mutant versions either alone or in the presence of Myc-DYRK2 were examined by WB. We show a representative experiment of three performed. The vertical lines indicate that the images are from different blots. D Extracts from cells expressing Flag-CDC25A-WT or the mutant version with eight Ser-to-Ala changes (Flag-CDC25A-8A), either alone or together with Myc-DYRK2 were analyzed by WB. We show a representative experiment of three performed. E Transfected HeLa cells with the indicated plasmids were treated with MG-132 (10 μM) for 12 h and lysed under denaturing conditions. Flag-tagged CDC25A-WT and the mutant version CDC25A-8A were purified by anti-Flag immunoprecipitation and ubiquitinated CDC25A detected by WB.