Figure 7

mr-s fused to GAL4 DNA binding domain functions as a transcriptional repressor in HEK293T cells. (A) Schematic drawing of the constructs used for the luciferase assay. 5xGAL4-pGL3 reporter plasmid was co-transfected into HEK293T cells with effector plasmids expressing various deletion mutants fused to GAL4-DBD. (B) Various amounts of DBD, DBD-mrs, DBD-N or DBD-C plasmids were transfected with 0.1 μg of 5xGAL4-pGL3 reporter plasmid. The reporter activity in the presence of the pcDNA3 vector (pcDNA3) was designated as 1. Error bars represent standard error of mean. (C) DBD-W404A and DBD-G453A were co-transfected into HEK293T cells with 5xGAL4-pGL3 reporter plasmid. Fold repression was calculated as the fold decrease in luciferase activity compared with DBD-mrs. Error bars represent standard deviation. (D) Various amounts of DBD-tail or DBD-SAM were transfected with 5xGAL4-pGL3 reporter plasmid. Error bars represent standard error of mean. (E) pcDNA3 or DBD-mrs (5 μg) was co-transfected into Y79 retinoblastoma cells with 0.5 μg of 5xGAL4-pGL3 reporter plasmid. The reporter activity in the presence of pcDNA3 was designated as 1. Error bars represent standard deviation. Asterisk marks statistically significant difference (Student's t test: p < 0.03). (F) Alignment of the C-terminal regions for mouse, rat, human, chick and zebrafish mr-s proteins. Conserved amino acid residues are shown with a dark shadow.