FIG. 3.
P52rIPK interacts with P58IPK in vivo. (A) Histidine reporter assay of yeast two-hybrid strains. Hf7c yeast strains harboring GAL4 AD and DNA BD plasmids expressing AD-PKR and BD-P58IPK (controls; position 1), AD-vector (AD-V) (control) and BD-P58IPK (position 2), AD-P52rIPK and BD-P58IPK (position 3), AD-P52rIPK and BD-vector (control; position 4), AD-GAL4 wild type (control) and BD-vector (position 5), and AD-vector and BD-vector (control; position 6) were replica plated in the presence (+ His) or absence (−His) of histidine, incubated for 5 days, and scored for growth. Growth on medium lacking histidine is indicative of a two-hybrid protein interaction. (B) Analysis of β-galactosidase activity. Strain Hf7c was cotransformed with expression plasmids encoding the indicated combination of AD and BD fusion proteins and patch-plated onto medium containing histidine in the presence of β-galactosidase substrate. After 3 days of growth, patches were scored for color development. Induction of the LacZ reporter (dark patch) is indicative of β-galactosidase activity and a two-hybrid protein interaction. The top row shows strains expressing AD-P52rIPK and (from left to right) BD-vector, BD-SV40 T antigen (BD-T ag); BD-lamin, and BD-P58IPK. The bottom row, left, shows strains expressing BD-P58IPK with AD-vector or AD-SV40 T antigen. The bottom row, right, shows strain Hf7c expressing the wild-type (wt) GAL4 protein. In this analysis and that shown in panel A, AD and BD fusion protein expression was confirmed by immunoblot analysis (data not shown).