Gfi1 is degraded by the ubiquitin-proteasome system. (A) HEK293 cells were split after transfection with Gfi1-GFP and were grown overnight with or without MG132. Cells were measured by flow cytometer and the mean fluorescence index (MFI) of untreated cells was set at 100%. After proteasome inhibition (5 μM MG132), a clear increase of Gfi1-GFP levels per cell was observed, indicated as MFI. (B) COS-1 cells were transfected with FLAG-Ub and Gfi1-GFP or with empty vector, followed by immunoprecipitation with an α-GFP antibody. Subsequent immunoblotting with an α-FLAG antibody resulted in the detection of a high molecular smear of proteins [(Ub)_n], indicating that Gfi1 is present in ubiquitinated complexes, or is ubiquitinated itself. → Indicates the height of unmodified Gfi1-GFP. Bottom panel with α-GFP staining of whole-cell extract (WCE) shows a clear distinct Gfi1-GFP band. (C) COS-1 cells were transfected with His-Ub and FLAG-Gfi1, and MG123 was added as indicated. Ubiquitinated proteins were selected with His-select beads under denaturing conditions, and immunoblotted for α-FLAG. Clearly visible are the ubiquitinated forms of Gfi1 [(Gfi1-Ub)_n], especially after proteasome inhibition. Whole-cell lysate was stained for FLAG, confirming equal loading. * Indicates α-specific binding of unmodified Gfi1 to His-select beads.