Fig. 2. Daxx deletion mutant transcriptional repression activity. (A) The indicated hDaxx deletion mutants (10 fmol), with domains indicated as described for Fig. 1, or the GAL4 DBD alone were co-transfected into NIH3T3 cells with a chloramphenicol acetyl transferase (CAT) reporter construct containing one GAL4-DNA-binding site. Forty-eight hours after transfection, CAT activity was determined as described in the Materials and Methods. All values were normalized for co-transfected secreted alkaline phosphatase activity and are presented as the percentage of CAT activity in the absence of hDaxx. Errors represent the standard deviation from four independent determinations. (B) The protein expression levels for the GAL4-hDaxx deletion mutants. Equivalent amounts of total cellular lysate from NIH3T3 cells overexpressing each of the individual GAL4-hDaxx deletion mutants (30 µg)were separated by either 10% SDS-PAGE (left panel) or by a 4-15% SDS-PAGE gradient gel (right panel). The level of protein expression was determined by western analysis using an anti-GAL4-DBD monoclonal antibody.