Figure 6. Comparison of the DNA-binding and transactivation properties of the proteins encoded by the two Nv-NF-κB alleles.

(A) An EMSA was performed using a radioactive κB-site probe and A293 cell extracts containing approximately equal amounts of each indicated FLAG-Nv-NF-κB protein (top panel). Two-fold dilutions of the equalized extracts spanning a 16-fold concentration range were used in the EMSA. The position of the NF-κB-DNA complex is indicated by the arrow. Anti-FLAG Western blotting (bottom panel) was performed to verify that the amounts of the two proteins were roughly equal. Probe, probe alone; Vector, an extract from pcDNA empty vector-transfected cells containing an amount of protein equal to the “16” lane of Nv-NF-κB-S67. (B) A reporter gene assay using a multimerized κB-site luciferase reporter gene was performed in A293 cells as described in Materials and Methods. Cells were co-transfected with expression plasmids either containing no insert (Vector) or the indicated Nv-NF-κB proteins. Values are relative to the normalized luciferase activity seen with the Vector (1.0). Values are the averages of three experiments, each performed with triplicate samples. An anti-FLAG Western blot of normalized extracts used in the reporter gene assay, to confirm approximately equal expression of the two Nv-NF-κB proteins (bottom panel).