Yeast two-hybrid analysis
The yeast two-hybrid reporter strain (EGY48) containing the DNA-binding domain (DBD) vector (pEG202)29 or the appropriate DBD fusion protein plasmids was transformed with the activation domain (AD) vector (pJG4–5)29 or the indicated AD fusion protein plasmids. Briefly, transformants were tested for a positive yeast two-hybrid interaction by expression of lacZ and galactose-dependent growth on media lacking leucine. Cells were grown at 30 °C on glucose or galactose minimal media, minimal media lacking leucine, or minimal media containing 200 μM 5-bromo-4-chloro-3-indolyl-β-d-galactosidase. The expression of the appropriately sized fusion proteins was confirmed by immunoblotting using either LexA antibody (1:1000 dilution, Covance) or HA antibody (1:1000 dilution, Santa Cruz).