ARR22 interacts with a subset of AHPs in the yeast two-hybrid assay. (A) Growth assay. Yeast clones expressing BD-ARR22 and the indicated AD clones were cultivated for 4 days at 28°C on either vector selective media (L-, W-) or interaction selective media (L-, W-, Ade-). The empty pGADT7 vector expressing the AD domain only was used as a negative control. (B) Quantitative β-galactosidase (LacZ reporter gene) activity assay. The specific β-galactosidase activity was measured in the extracts of three independent yeast clones expressing BD-ARR22 and the indicated AD fusion protein. Background activity (white bars) was determined for extracts from clones not growing on interaction selective media. Extracts from yeast clones showing above-background activity and demonstrating in vivo interaction are indicated in grey. Data are expressed as mean +/- SD (n = 3). (C) Western-blot detection of the fusion proteins expressed in the yeast cells used for the two-hybrid analysis. The immunodetection of the BD-ARR22 was carried out with an antibody directed against the c-myc tag (anti c-myc). The AD fusion proteins were detected with an antibody directed against the HA tag (anti HA). 15 μg of total protein were loaded per lane.