Analysis of prestin-bait expressing yeast. (A). Expression of the mPrestin-Cub-LexA-VP16 bait fusion protein (~120 KDa) in yeast was verified by SDS-PAGE/Western blot analysis using anti-prestin. (B). Both negative and positive control prey proteins were expressed in prestin-bait yeast as demonstrated by their growth on the SD-LT plate. Prestin interacted with the positive control prey (NubI), as indicated by its growth on the SD-LTH plate, but not with the negative control prey (NubG). These data suggest that prestin-protein bait is expressed in the correct orientation with the Cub-LexA-VP16 accessible to the NubG tag of the prey protein and that NubG is not able to reconstitute ubiquitin with mPrestin-Cub-LexA-VP16.