Figure 2.

The first ankyrin repeat of IκBα is sufficient to convert IκBβ and IκBɛ from weak to strong inhibitors of NF-κB activity in vivo. (A) P19 cells were transfected with the PRDII₄CAT reporter plasmid (100 ng) along with a constant amount of CMV p65 expression vector (100 ng) and increasing amounts of the indicated IκB hybrids (10 ng, 30 ng, 100 ng, 300 ng, 1000 ng, and 3000 ng). The resulting CAT activities were plotted as a fraction of the IκB expression vector amount used in the transfection. The inhibition index was derived as described in Materials and Methods by averaging the results obtained from six independent experiments. The variability from experiment to experiment was less than 20%, whereas the variability between individual constructs in the same experiment was less than 5%. (B) Shown is a Western blot using whole cell extracts derived from cells transfected with the indicated IκB expression vectors that were immunoblotted with IκBα (lanes 1–5, 9–10), IκBβ (lanes 6–8), or IκBɛ (lanes 11–13), specific antibodies. All IκB hybrids display the expected size, indicating thus their integrity.