![Figure 2. 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.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/109/1/10.1182_blood-2006-02-003590/4/zh80010705870002.jpeg?Expires=1735557683&Signature=ZtLYVMq1fFxFfjzKIvydhENm5NvCJJmoFMKRvhJI2szsJnmAMp6YJfUxjT7YY48i4SrzU4usbwarF21BXwWnjLfknKYMVJGX5TuIkULUIQNgtHdsrFjOh~ty7NCis5fVTO614XGyCG3jfJk1J7kIAfsJ44c6L~TNxWaw0RLowVj8Z9NDD-OoXthQ1TIe5aJWu4~3jtY-6l8A6d6QSDgztMHr5agp0gf5HJ7wob2sLoxyZNVft6ujbXgm1Qr1eAj-xM3bAseDqSN-Cg7lMhASNslhUl1mAoO2m2Vt8TSZLTo06wT6~Mdp1qw9J16EDiamFAxPquytZotN7XNLGULI1A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
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.
