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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: CK2-mediated CCDC106 phosphorylation is required for p53 degradation in cancer progression

Fig. 3

Phosphorylation of CCDC106 at S130 and S147 is required for CCDC106 interaction with p53. a The subcellular localization of wild-type and mutant EGFP-CCDC106 fusion proteins. The EGFP fusion protein-expressing plasmids were transfected into HeLa cells, and EGFP fluorescence was examined directly at 24 h posttransfection by fluorescence microscopy. Nuclei were stained with Hoechst 33258. Merge represents the combined image of EGFP fluorescence and nucleus staining. b The influence of CX-4945 treatment on the subcellular localization of the CCDC106 protein in HeLa and MCF7 cells. At 24 h after treatment with different concentrations of CX-4945, cell lysates were harvested, and the cytoplasmic (C) and nuclear (N) fractions were separated and subjected to WB. PCNA and ACTB were used as markers of nuclear and cytoplasmic proteins, respectively. c The interaction between p53 and wild-type or mutant p53 was analyzed by a Co-IP assay. HEK293 cells were transiently transfected with HA-p53 and Myc-tagged wild-type or mutant CCDC106 expression plasmids. Cell lysates (800 μg) were precipitated with an IgG or anti-Myc antibody, and the immune complexes were subjected to WB with an anti-HA or anti-Myc antibody. The input is equivalent to 10% of the lysate used for co-IP. d The interaction between p53 and wild-type or mutant p53 was detected by a GST pull-down assay. The bacterially expressed and purified GST-p53 fusion protein was incubated with the total lysates of HeLa cells transfected with a wild-type or mutant CCDC106 expression plasmid. GST-p53 was pulled down with glutathione agarose beads, and the associated Myc-CCDC106 fusion protein was analyzed by Western blotting with an antibody against Myc-tag. λ: cell lysate treated with λ protein phosphatase

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