Figure 5.

Ira1 and Ira2 function independently of Mad2 in the spindle assembly checkpoint (SAC) pathway. (A) Δira1Δira2Δmad2 was highly sensitive to spindle damage. Wild-type, Δira1Δira2 (strain YSK2866), Δmad2 (strain YSK2668), and Δira1Δira2Δmad2 (strain YSK2925) cells were grown to mid-log phase, serially diluted 10-fold, spotted onto either YPAD plates or YPAD plates containing 10 μg/mL benomyl and then incubated at either 25°C or 30°C. (B) Δira1Δira2Δmad2 cells are more sensitive to nocodazole than Δira1Δira2 or Δmad2 cells. Wild-type, Δira1Δira2, Δmad2, and Δira1Δira2Δmad2 cells expressing Myc-tagged Pds1 (strains YSK2992, YSK3001, YSK2984, and YSK3003, respectively) were grown to early log phase at 25°C and then treated with 15 μg/mL nocodazole for 130 min. Cells were collected every 10 min from 60 min, and then Pds1 expression (Pds1-9Myc) was detected by western blotting using an anti-Myc antibody. α-Tubulin (Tub1) served as a loading control.