Figure 6

CtBP1 has a role in the stimulatory effect of E1A mutants on the K18 promoter. (A) CtBP1 interacts with E1A-N190 but not with the mutant E1A-N190-mut 281-282. GST pull-down assays were performed on extracts of SW613-3 (3) or SW613-B3 (B3) cells using GST alone (empty vector) or the fusion proteins GST-E1A-N190 or GST-E1A-N190-mut281-282. Bound proteins were analyzed by Western blotting with an anti-CtBP1 antibody. (B) Effect of the E1A-N190 or E1A-N190-mut281-282 proteins on the activity of the K18 promoter. SW613-3 (T) or SW613-B3 (NT) cells were transfected with plasmid pK18(80)luc alone (none) or in the presence of plasmid pE1A-N190 or pE1A-N190-mut281-282 as indicated. Results are presented as in figure 3. Error bars represent standard deviation of the mean of duplicate experimental points. (C) Reversion of the stimulatory effect of E1A-N190 by CtBP1. SW613-3 (T) or SW613-B3 (NT) cells were transfected with plasmid pK18(80)luc alone (none) or in the presence of plasmid pE1A-N190 (2 μg) or pcDNA3-CtBP (24 μg) or with the three plasmids together, as indicated. Results are presented as in figure 3. Error bars represent standard deviation of the mean of triplicate experimental points. (D) The CtBP1 protein has no direct effect on the activity of the K18 promoter. SW613-3 (T) or SW613-B3 (NT) cells were transfected with plasmid pK18(80)luc alone (none) or in the presence of various amounts of plasmid pcDNA3-CtBP, as indicated. Results are presented as in figure 3. Error bars represent standard deviation of the mean of triplicate experimental points.