Skip to main content
Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: CK2-mediated phosphorylation of Che-1/AATF is required for its pro-proliferative activity

Fig. 3

Che-1 phosphorylation is required for its pro-proliferative ability. A Cell proliferation analysis of HCT116 cells transiently transfected with Myc-Che-1, Myc-Che-1 3S or control vector (pCS2MT). Bar plot shows the average number of cells observed in these experiments (n = 6). B Representative WB analysis with the indicated antibodies of total cell extracts from HCT116 cells transfected as in A. C 5-FUrd labelled RNA was evaluated by immunostaining with anti-BrdU antibody in HeLa cells transiently transfected with the indicated expression vectors. Nuclei were stained with Hoechst dye. Scal bar, 10 μm (left). Bar plot showing the percentage of 5-FUrd positive nuclei (right, top). Percentage was calculated from 100 randomly selected cells for each group. Representative WB showing the transfection efficiency of the 5-FUrd incorporation assay described above (right, bottom). D WB analysis with the indicated antibodies of total cell extracts from HCT116 cells transfected as in A. E WB analysis of total cell extracts of HCT116 cells transfected with siControl or siChe-1 3’UTR alone or in combination with empty vector or with Che-1 expressing vectors (Myc-Che-1 and Myc-Che-1 3S). F Nuclear extracts from HCT116 cells transiently transfected with Myc-Che-1 wt or 3S mutant, were subjected to IP with anti-Myc monoclonal antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies. Input corresponds to 10% of the nuclear extracts used for IP (left). Relative H3 binding was calculated from three different experiments by densitometry. H3 intensity was normalized to the one of Myc-Che-1 wt or 3S mutant (right). G WB analysis of GST-pull down experiment conducted using total extracts from HCT116 cells over-expressing pCI-HDAC 1 incubated with purified GST-H3 fusion protein or control GST agarose beads, in presence or absence of the indicated peptides. GST fusion proteins expression is shown by Comassie blue staining. H MNase digestion pattern of nuclei (left) and corresponding WB analysis (right) with the indicated antibodies of total cell extracts from HCT116 cells transfected as in A. Statistical significance is indicated by asterisks as follow: *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, n.s. = not significant

Back to article page