Figure 4: Bfa1 phosphorylation profile during spindle misalignment.

(a,b) Cells were released from G1 (time-point zero) arrest under dyn1-IAA17 depleting conditions and samples were collected at indicated time points for immunoblotting and microscopy. (a) Immunoblots showing Bfa1-GFP mobility shift during spindle misalignment. Clb2 and Sic1 served as markers for cell-cycle progression. Arrows point to the hyperphosphorylated forms of Bfa1. (b) The graphs show the percentage of cells with misaligned nuclei per time point. Samples were analyzed by microscopy after DAPI staining. (c,d) Immunoblots showing Bfa1-GFP mobility shift during anaphase arrest, in temperature-sensitive cdc5-10 cells at restrictive temperature (c) and in cells where Tem1 or Cdc5 were depleted (d). The 100 min sample of kin4Δ cells from the experiment shown in (a) was loaded as a reference for mobility shift in (c). Arrows point to the hyperphosphorylated forms of Bfa1. (e,f) Effect of artificial Cdc5 recruitment to the SPBs on Bfa1 phosphorylation profile and on SPOC integrity. (e) Immunoblot showing Bfa1-3HA mobility shift in logarithmic growing cultures of Cdc5-GFP cells, in the presence or absence of SPC42-GBP and SPO12. Arrows point to the hyperphosphorylated forms of Bfa1. (f) Percentage of SPOC-deficient phenotypes in indicated cells types. Graph is an average of three independent experiments. Error bars show s.d. Per experiment, 100 cells were counted per strain. Asterisk indicates significant difference of the indicated sample from other samples, according to two-tailed Student’s t-test (P<0.05).