FIG. 1.

WT PITX2a can form dimers. (A) The amino acid numbering for the schematic corresponds to the PITX2a isoform. The black region represents the homeodomain (HD). Mated yeast culture (5 μl) was spotted on SD/−His/−Leu/−Trp plates with 0 to 10 mM 3-AT (−HIS) and SD/−Ade/−His/−Leu/−Trp plates (−HIS −ADE) and incubated at 30°C for 5 days. The SD/−Leu/−Trp auxotrophic medium was used in all plates to maintain bait and prey selection. The β-galactosidase activity was assayed from mated yeast colonies by using chemiluminescent detection. The vector interaction with the PITX2a bait or prey was set to 1. The data represent results from four to six separate colonies obtained from two to three separate matings and represent the mean β-galactosidase activity (relative to that of vector) ± the standard error of the mean. (B) Schematic representation of the PITX2 constructs used as baits in the yeast-two hybrid analysis. The mated yeast cultures were spotted on −HIS plates with 0 to 2.5 mM 3-AT and −HIS −ADE plates; β-galactosidase activity was measured as described in the legend for panel A. The data represent results from four to six colonies from two to three separate matings. ND, not determined. (C) The expression of bait constructs used in panels A and B were confirmed by Western blotting by using the anti-GAL4 DBD antibody. Full-length PITX2a bait and molecular weight markers are indicated.