FIG. 1.

Screening of cDNA libraries using Eed as a bait in yeast two-hybrid system. (A) cDNA libraries from mouse embryos 9.5 to 10.5 days postcoitum or Jurkat cells were screened in yeast against the LexA-Eed bait construct as described in Materials and Methods. The cDNA plasmids from primary positive clones were purified and transformed into yeast strains carrying either LexA-Eed wild-type or LexA-T1040C mutant Eed bait. Three colonies from each transformation were grown on plate with selective media (Leu⁻, Trp⁻) and then transferred onto nitrocellulose filter and assayed for β-galactosidase activity. The blue color was developed for 3 h at 30°C. (B) The transformants were also checked for expression of the bait constructs. Cells transformed with pBT116 (Vector), LexA-Eed, and LexA-T1040C plasmids were grown overnight in selective medium, lysed in SDS sample buffer, electrophoresed, transferred onto a polyvinylidene difluoride membrane, and stained with anti-LexA rabbit serum followed by anti-rabbit immunoglobulin G-alkaline phosphatase conjugate. A membrane stained with 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium phosphatase substrate is shown.