Fig. 2
From: The FBXW7-binding sites on FAM83D are potential targets for cancer therapy
The importance of residues H343/L344 of FAM83D binding to FBXW7 for FAM83D-mediated down-regulation of FBXW7. A, The protein levels of FBXW7 and its downstream substrates including Cyclin E, Aurora A and c-Myc were detected by western blotting after FAM83D WT and H343R/L344A mutant (M2) were reintroduced into MCF7 cells. B, The effect of FAM83D H343R/L344A mutant (M2) on the ubiquitnation of FBXW7 was determined by co-immunoprecipitation analysis. HA-tagged FBXW7 was co-transfected into HEK293T cells with Myc-tagged ubiquitin (My-Ub) and Flag-tagged wild-type FAM83D (Flag-FAM83D WT), or Flag-tagged FAM83D mutant (Flag-FAM83D M2). Immunoprecipitation with anti-HA antibody followed by anti-Myc antibody immunoblotting. C, The effect of FAM83D H343R/L344A mutant (M2) on FBXW7 protein stability was determined by cycloheximide (CHX) chase assay. MCF7 cells were respectively transfected with FAM83D WT or H343R/L344A mutant (M2) followed by treatment with 50 µg/mL CHX for the indicated time intervals. Endogenous FBXW7 protein degradation was detected by western blotting. The graphs show quantitative analysis of CHX chase data. Data were presented by mean ± SD of three independent experiments. Ns: not significant. *: p < 0.05 based on the Student’s t-test