Figure 10

Investigation of transcriptional repression using a panel of GAL4-PHF1b fusion proteins. A) Full length PHF1b and truncated PHF1b versions fused to GAL4 (1–100) were recruited upstream of the TK enhancer promoter which was linked to a luciferase reporter gene (pG5-200tkLUC). GAL4 (1–100) contains the DNA binding domain that recognizes GAL4 regulatory sites. GAL4-PHF1b fusions were expressed from a CMV promoter. COS-7 cells were co-transfected with the reporter plasmid (pG5-200tkLUC) and the constructs as indicated. 48 hours after transfection, cells were harvested and assayed for luciferase activity. Results shown are mean values ± SEM and normalized to protein content within each dish as well as to vector control (pG5-200tkLUC+ GAL4 (1–100) defined as 100%). “*” indicates significantly different from vector control (p < 0.05) as determined by 95% confidence interval. B) GAL4-PHF1b fusion proteins are expressed in COS-7 cells as shown by Western analysis. Extracts of COS-7 cells mock transfected or expressing PHF1b constructs were analyzed by Western analysis using a GAL4 antisera. No proteins were detected in mock-transfected cells (1) and proteins were detected for cells transfected with PHF1b (2), PHF1bΔ4 (3), PHF1bΔ3 (4), PHF1bΔ6 (5). Molecular size markers are to the right.