FIG 5.

Fbw7 regulates endogenous the Eya1 ubiquitination level and half-life. (A and B) Flag/Eya1, HA/UB, and Myc/Fbw7 were coexpressed in HEK293 cells (A) or FBW7 knockout HCT116 cells (B), and the Flag/Eya1 ubiquitination level was analyzed using the indicated antibodies in an IP/IB assay. (C) Endogenous EYA1 protein levels in wild-type and FBW7^−/− HCT116 cells. (D to G) Flag/Eya1 and variants were expressed in wild-type (D and E) and FBW7^−/− (F and G) HCT116 cells. Cells were treated with cycloheximide (CHX) (20 μg/ml), and the Flag/Eya1 protein level was chased in a period of 6 h and quantified using ImageJ based on signal density and a protein loading control (GAPDH).