Comparing kinesin-5 expressi on and spindle localization. (A) Example of western blot against Cin8-3HA (121 kDa) and Kip1-3HA (130 kDa) or Zwf1/Glucose-6-phosphate dehydrogenase loading control. Cells were arrested in G1, released, and the lysate was collected every 15 min. (B) Quantification of time course western blots against Cin8-3HA and Kip1-3HA such as in panel A. Anti-HA intensity normalized to Zwf1 loading control and then normalized to total anti-HA signal at 120 min. Error bars are standard deviation. n = 2 independent experiments; *P < 0.05 by Student’s t test. (C) Example montage of a cell expressing Cin8-3GFP (green) and Spc110-tdTomato (magenta). Arrowheads point out Cin8 localization adjacent to the spindle poles during late anaphase. First time point is 10 min prior to anaphase onset. Time interval = 2 min. Scale bar = 1 µm. (D) Quantification of background-subtracted Cin8-3GFP spindle fluorescence intensity (left axis) and spindle length (right axis) as a function of time since anaphase onset. Cin8-3GFP intensity normalized to anaphase onset, which is indicated by the dashed vertical line. Error bars are mean ± 95% CI. n = 21 cells. (E) Example montage of a cell expressing Kip1-mNeonGreen (green) and Spc110-tdTomato (magenta). Arrowhead points out Kip1 localization at the middle of the spindle during late anaphase. First time point is 10 min prior to anaphase onset. Time interval = 2 min. Scale bar = 1 µm. (F) Quantification of background-subtracted Kip1-mNeonGreen spindle fluorescence intensity (left axis) and spindle length (right axis) as a function of time since anaphase onset. Kip1-mNeonGreen intensity normalized to anaphase onset, which is indicated by the dashed vertical line. Error bars are mean ± 95% CI. n = 24 cells. Source data are available for this figure: SourceData F2.