FIGURE 7.

SiKAT2 selective interact with SiSNARE proteins. Yeast mating-based split-ubiquitin assay for the interaction of SiSYP121, SiVAMP721, and SiSNAP33 with SiKAT2-Cub as baits. After several attempts, SiKAT3 failed to transform into yeast. Yeast diploids were created with NubG fusion constructs of each of the SiSNARE proteins together with controls (NubG, negative; NubI, positive) spotted (left to right) on CSM medium without Leu, Trp, Ura, and Met (CSM_–TULM) to verify mating, CSM medium without Leu, Trp, Ura, Met, Ade, and His (CSM_–AHTULM) to verify adenine- and His-independent growth, and on CSM_–LTUMAH with the addition of Met to verify interaction at lower K⁺ channel-Cub expression levels. Diploid yeast was dropped at 1.0 and 0.1 OD600 in each case. Immunoblot analysis (5 μg total protein/lane) of the haploid yeast used for mating (right) using commercial α-HA antibody for the SiSNARE fusions and α-VP16 antibody for the K⁺ channel fusions.