FIG. 6.

Sequential induction of IκBα and GFP-p65. (A) HA-IκBα gene was cloned into an expression vector that contains a copper-inducible promoter. GFP-p65 was expressed from the galactose-inducible promoter. Double transformants in different yeast strains were treated first with copper to induce IκBα expression and then with galactose to induce GFP-p65 expression. Whole-cell extracts were prepared from double transformants that were not treated either with inducing agent (lanes 1, 4, and 7), or were treated with 0.5 mM copper sulfate for 1 h (lanes 2, 5, and 8) or with copper sulfate for 1 h followed by galactose and copper for an additional 2.5 h (lanes 3, 6, and 9). GFP-p65 and IκBα were detected by immunoblotting after separation of the extracts by SDS-PAGE. crm1-1 and CRM1⁺ strains were defined in the legend to Fig. 1; W303 represents another WT strain. Results shown are from one of three independent experiments. (B and C) Fluorescent visualization of GFP-p65 localization in single and double transformants, respectively, as noted on the left of the panels. p424 is an empty expression vector. Yeast strains used are indicated on the top. Results shown are from one of three independent experiments. (D) Nuclear association of GFP-p65 and HA-IκBα in crm1-1 cells. CRM1⁺ and crm1-1 cells transformed with expression vectors described for panel A were induced (+) to express GFP-p65 alone, HA-IκBα alone, or both together as indicated. Whole-cell extracts were first incubated with anti-IκBα antibodies, and then the immunoprecipitate was fractionated by SDS-PAGE. Proteins were transferred to nitrocellulose filters which were probed with anti-p65 and anti-IκBα anti-sera. The immunoblots were visualized by chemiluminescence.