Structural requirement for GATA-1 mediated PU.1 repression.

(A) The structure of the chicken GATA-1 protein and mutant proteins used in this study. The 2 zinc fingers are shown as boxes; numbers indicate amino acids. Mutated zinc fingers are shown in black. (B) The pPU3-TK-LUC reporter (100 ng), pCMV-MTPU.1 expression vector (0.25 μg), and pRSV-βgal (100 ng) internal control plasmids were cotransfected into Q2bn cells with 1 μg of pSPCMV-GATA-1 (WT) or the corresponding expression vectors for the mutant GATA-1 proteins as in Figure 3. The fold repression relative to the value obtained by cotransfection of pCMV-MTPU.1 in the absence of GATA-1 was calculated after normalization of the luciferase values to the β-galactosidase activity. The data shown are the average of 2 determinations from a representative experiment. (C) The expression of the wild-type and mutant GATA-1 proteins in the Q2bn cells transfected in (B) was analyzed by Western blotting as described in Figure 3B. Bands corresponding to the undegraded GATA-1 proteins have been marked with an asterisk (*).