Specific activation of the c-kit promoter by a multifactorial complex containing SCL, E47, LMO2, Ldb-1, and GATA factors (SCL complex).

(A) NIH 3T3 cells were cotransfected with the kit-1146 reporter and expression vectors encoding SCL (60 to 1500 ng), E47 (150 ng), LMO2 (750 ng), Ldb-1 (60 to 1500 ng), and GATA factors (150 ng). The plus and minus signs indicate, respectively, inclusion or omission of specific expression vectors in the transfection mixtures. Open bar: reporter construct alone. (B) Coexpression of transfected vectors was confirmed by Western blotting. Nuclear extracts of TF-1 cells (12.5 μg) and of NIH 3T3 cells (12.5 μg), transfected with the indicated expression vectors, were immunoblotted with the antibodies shown on the left of each panel. Arrowheads indicate specific bands. The upper band in the Ldb-1 immunoblot corresponds to a cross-hybridizing protein. (C) The SCL complex does not activate the c-fms, G-CSFR, or minimal SCL (SCL-44) promoters, nor does it affect the activation of the G-CSFR promoter by C/EBP-α (CCAAT/enhancer binding protein α; 150 ng). Each reporter plasmid was transfected in NIH 3T3 cells in the absence (open bars) or presence (solid bars) of the SCL complex. For panels A and C, results are shown as luciferase activity relative to the empty pXPIII vector (on average, 2000 relative light units [RLUs]), represent the average ± SD of replicate determinations, and are representative of 8 and 3 independent experiments, respectively. Luciferase reporter activities were normalized to that of an internal control (CMV-βgal).