GFI1^36S and GFI1^36N proteins show different subnuclear localization. (A) NIH-3T3 cells were transfected with GFI1^36S expression plasmid. GFI1 appears green; DNA, blue. The right column represents the merging of both staining. The numbers of cells analyzed for GFI1^36S are indicated. GFI1^36S is mainly localized in a dotted pattern. (B) NIH-3T3 cells were transfected with a GFI1^36N expression plasmid. The numbers of cells analyzed for GFI1^36N are indicated. GFI1^36N is localized at the nuclear border. (C) NIH-3T3 cells were transfected with either a GFI1^36S or GFI1^36N expression plasmid. Cell lysates were fractionated. Both protein variants are localized in the nuclear cell fraction and cannot be found in the cytosolic fraction. (D) Analysis of a bone marrow sample from the only available homozygous for GFI1^36N. As in transfected cells, GFI1^36N was mainly located at the nuclear/cytoplasmic border. As a control, cells from the Kasumi 1 t(8;21)-positive AML cell line were used. This cell line was originally derived from a patient with French-American-British M2 AML and is homozygous for GFI1^36S. (E) Nuclear matrix preparation of NIH-3T3 cells transiently transfected with expression vectors for GFI1^36S or GFI1^36N. Both variants are still attached to nuclear matrix components, although the GFI1^36N variant appears to be more concentrated at the nuclear membrane, consistent with our observation in regular cell transfection assays.