FIG 2.

GFI1 is SUMOylated and ubiquitinated. (A) High-molecular-weight (HMW) derivatives of GFI1 identified under instantly denaturing conditions. COS7L cells were transfected with empty vector or expression constructs for Flag-tagged GFI1, HDAC1, and β-catenin. Cells were collected in-ice cold PBS, snap-frozen in liquid nitrogen, and suspended in 20% TCA. Precipitated proteins were solubilized by boiling in solubilizing buffer. Aliquots were analyzed by immunoblotting with anti-Flag antibody M2. (B) HMW derivatives of GFI1 contain SUMO and ubiquitin modifications. Flag-tagged GFI1 was expressed in COS7L cells, collected, and processed as described for panel A. Anti-Flag immune complexes (IC) and whole-cell lysate (WCL) were analyzed by SDS-PAGE and Western blotting (WB) as shown. (C) Each SUMO paralog can conjugate GFI1. Flag-tagged SUMO1, SUMO2, or SUMO3 was expressed in COS7L cells with myc-tagged GFI1 as shown. Cells were harvested as described for panel A. SUMOylated proteins were collected by anti-Flag immune precipitation. Immune complexes and whole-cell lysates were analyzed by Western blotting (WB). (D) Endogenously expressed GFI1 is SUMOylated. HL-60 cells in the quantities shown, stably expressing Flag-tagged SUMO2, SUMO3, or vector control, were collected and processed as described for panel A and then immunopurified via the Flag epitope tag (FL). Immune complexes (IC) were probed for GFI1 by Western blotting (WB). Expression of GFI1 and SUMO proteins was confirmed in whole-cell lysates (WCL) by Western blotting. Ub, ubiquitin.