Pairwise interactions
Specific interactions were tested by transforming competent MaV203 yeast cells with one bait construct (in pDBLeu) and one prey construct (in pPC86) and testing the resulting colonies for activation of the HIS3, URA3 and LacZ reporters as before.
To create a full-length Homer3 clone for pairwise tests, a cDNA clone (IMAGE clone 3602414, accession number BE569374) containing the missing N-terminal 70 amino acids was identified by a BLAST search of the EST nucleotide sequence database and obtained from the Rosalind Franklin Centre for Genomics Research. The missing fragment was amplified from the IMAGE clone by PCR and inserted into the pPC86-Homer3 plasmid to create a full-length expression construct.