Fig. 5

CHD4 protein stability is regulated by FBXW7-mediated ubiquitination. A Sequence alignment of the phosphodegron sequences recognized by FBXW7 in CHD4. Conserved degron sequences are shown in red. B Scatter diagram showing the correlation between the RNA expression levels of CHD4 and FBW7 in TNBC patients based on TCGA-BRCA-TNBC datasets. P values are indicated. C Representative IHC images of FBXW7 and CHD4 staining. The scale bar indicates 50 μm. D The correlations between the protein expression of CHD4 and FBXW7 in TNBC tissues. E Western blot analysis of the regulation of CHD4 and JUN protein expression by FBXW7 in two transfected TNBC cell lines. F Western blot analysis of CHD4 protein and HA-tag protein in HCC1937 cells with different treatments. which was indicated in the figure labels. G MDA-MB-231 cells were transfected with shNC or shFBXW7 plasmid and evaluated for CHD4 protein half-life by CHX chase assay. H MDA-MB-231 cells were transfected with Flag-CHD4 or Flag-CHD4 ∆TPXXS plasmids and evaluated for CHD4 protein half-life by CHX chase assay. I Western blot analysis of CHD4 protein and HA-tag protein in HCC1937 cells transfected with HA-FBXW7 or HA-FBXW7 ∆Fbox plasmid. J MDA-MB-231 cells were infected with the indicated constructs for 48 h. Cells were subjected to Western blot analysis. K Western blot analysis of Flag-tag protein and HA-tag protein in HCC1937 cells transfected with indicated plasmid. L MDA-MB-231 cells were transfected with empty vector, HA-FBXW7 or HA-FBXW7∆Fbox plasmid and evaluated for CHD4 protein half-life by CHX chase assay. GAPDH served as an internal reference in all of the above Western blot analyses