Fig. 6. The localization of Mob2p and Orb6p is interdependent. (A) Wild-type (YDM1203) and orb6-25 mutant cells (YDM1204) expressing Mob2p-GFP were grown to exponential phase at 25°C and then shifted to 36°C for 5 hours. GFP fluorescence and DIC images of live cells are shown. (B) Mob2p levels in orb6-25 mutant cells. Wild-type (YDM975) and orb6-25 mutant cells (YDM1015) expressing Mob2p-13Myc were grown as in A, and Mob2p-13Myc levels were analyzed by western blotting with antibodies against Myc. The lower band is IgG(H). (C) Orb6p-3HA level in mob2 shut-off strain. The mob2 shut-off strain expressing Orb6p-3HA (YDM1080) was grown to exponential phase at 30°C in the absence of thiamine and then grown in the presence of 5 µg/ml thiamine for 16 hours and 22 hours. Orb6p levels were determined by western blotting with antibodies against the HA epitope. Cdc2p was used as a loading control and detected by anti-PSTAIRE antibody.