Yeast mating to identify TWIST protein interactions. (A) Yeast cells transformed with the indicated combinations of bait (pGBKT7-TWIST) and prey (pGAD-TCF-4) plasmids on YPD plate. Co-transformed yeasts were spotted on -Leu-Trp plate representing the successful cell mating (irrespective if fusion proteins interact); by colony lift assay. TWIST and TCF4 protein interaction was visualized on selective QDO Plates which contains -Leu-Trp-Met-His (quadrate drop out) reporter plates containing 1 mM 3-aminotriazole (3-AT). In this assay, p53/pGBKT7 clone transformed into AH109 mated with Y187 strain containing T7-antigene in pGADT7-vectors was used as a positive control and p53/pGBKT7 in AH109 mated with LamC/pGADT7 which was transformed into Y187 used as a negative control to confirm specificity of the interaction. (B) NLS rescue assay to study the ability of TCF-4 to assist in the nuclear localization of TWISTNLS1 mutants. Co-transfection of K38R, K73R, and K77R with TCF4 results in the restoration of TWIST mutants in the nucleus.
Singh and Gramolini BMC Cell Biology 2009 10:47 doi:10.1186/1471-2121-10-47