Gfi1 is degraded by the 26S proteasome in hematopoietic cells. (A) The DNA binding activity of in vitro–translated Gfi1 was checked using EMSA after incubation with 32 P-labeled oligos containing a consensus Gfi1 binding site. A clear band shift (→) was found only in the lanes containing 1 μL or 3 μL Gfi1-programmed reticulocyte lysate. Unprogrammed reticulocyte lysate was used as negative control (NC). (B) An in vitro degradation assay using 35S-labeled in vitro–translated Gfi1 incubated with U937 lysates at 37°C in the presence of vehicle, or the proteasome inhibitors MG132 or Velcade or denatured cell lysate (5 minutes at 100°C). At the indicated time points, samples were inactivated in loading buffer and resolved by SDS-PAGE. (C) The localization of 2 putative PEST domains in Homo sapiens Gfi1 is underlined. (D) 35 S-labeled in vitro–translated full-length Gfi1, the zinc finger domain of Gfi1 (ZN Gfi1), and the non–zinc finger domain of Gfi1 (ΔZN Gfi1) were used in an in vitro degradation assay as described in panel B.
