The SNAG domain of Gfi-1B is indispensable for induction of erythroid differentiation. (A) Schematic representation of the different constructs cloned into the retroviral vector MIGR. Full-length Gfi-1B (Gfi-1B), Flag-tagged at the N-terminus (NFlag) or at the C-terminus (CFlag), SNAG-deleted cDNA without (Δ1) or with (Δ1CFlag) at the C-terminus. (B) Immunofluorescence performed with an anti-Flag antibody showing a similar nuclear expression of all tagged proteins. DAPI staining reveals nucleus, all 4 populations are GFP positive, and anti-Flag antibody reveals Gfi-1B–infected cells. Cells were examined under a Nikon Eclipse E600 epifluorescence microscope equipped with an Apochromat Plan 60 ×/1.4 objective lens (Nikon, Tokyo, Japan). Images were acquired with a CoolSnap camera (Photometrics, Evry, France) and processed with Adobe Photoshop 7.0 software (Adobe, San Jose, CA). (C) Analysis of erythroid differentiation induced by Gfi-1B–forced expression. UT7 and K562 were infected with the empty MIGR or with the MIGR carrying the N-(Nflag) or the C-(Cflag) Flag or the deleted SNAG (Δ1) cDNA. GPA expression was determined by flow cytometry, 4 days after retroviral infection in UT7 cells (left). Hemoglobinization was determined by benzidine staining 6 days after infection of K562 cells (right). Data are expressed as mean ± SD.