Figure 5

Effects of the RH2ED mutation on interaction with NH1 and NH1-mediated transcriptional activation. (A) Protein-protein interaction in yeast two-hybrid. Yeast two-hybrid tests were done as described in Figure 3. (B) Western analysis of yeast expressed fusion proteins. Protein was extracted from yeast cells containing constructs expressing LexA:NH1 plus B42AD (labeled B42), B42AD:NRR (NRR), B42AD:RH1 (RH1), B42AD:RH2 (RH2), B42AD:RH3 (RH3), or B42AD:RH2ED (RH2ED). Extracted protein samples were run on a 5-20% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was probed with anti-HA and anti-LexA antibodies sequentially. Protein loading was normalized to amount of input yeast cells. (C) Effects of the RH2ED mutation on NH1-mediated transcriptional activation. The protoplast transient assay was done as described in Figure 1. (D) Western analyses of transiently expressed proteins in protoplasts. Protoplast transfection and protein preparation were done as described in Figure 4. Protein on a nitrocellulose membrane was probed with an anti-HA antibody. A duplicate membrane was probed with anti-NH1 and anti-Gal4DB antibodies sequentially. The amount of protein loaded was normalized to the Gus activity.