Characterization of EKLF-ER™–transduced cell lines.

(A-C) Analysis of clonality and expression in GFP⁺cell lines. (A) Southern blot of genomic DNA from the parent EKLF^−/− line A2 and GFP⁺ subclones (named A2.1 through 6). The probe used is described in the legend for Figure2A. The endogenous 8.8-kb EKLF BamHI fragment (situated 5′ of the region deleted in the targeting construct) is indicated. Each cell line has a least one band of alternative size corresponding to a unique site of proviral integration, indicating independent clonality in each case. (B,C) Western blots for EKLF in whole-cell lysates made from sublines derived from the parent lines B1 (B) and A2 (C). The blots were probed with an antibody specific for EKLF (top) or, after stripping, for GATA-1 (bottom). Mock and EKLF represent extracts from nontransfected 293T cells and 293T cells transfected with an expression vector designed to express a glutathione-S-transferase–EKLF fusion protein, respectively. Migration of molecular-weight makers is indicated on the left side. The position of EKLF-ER™ and GATA-1 are indicated. (D-G) Nuclear translocation of EKLF on stimulation with 4-hydroxytamoxifen. Immunofluorescence staining of B1.4 cells for EKLF (Cy3 [red]; E,G) and DAPI (blue; D, F) after 72 hours of treatment with 100 nM 4-hydroxytamoxifen (F,G) or ethanol as a vehicle control (D,E). The overlaid line in panel D shows the boundary of the nucleus of a single large cell. The outer circle in panel E shows the cytoplasmic boundary of the same cell after excitation of Cy3 (EKLF). Most detectable EKLF protein shifted from the cytoplasm (area between the 2 circles in panel E) to the nucleus (G) after 72 hours in tamoxifen.