In vitro microtubules from whole-cell lysate and Cin8 minus-end velocity. (A) Example images of microtubules assembled in vitro from whole-cell lysate from cells expressing GFP-Tub1 before and after high salt washes. Scale bars = 10 µm. (B) Example images of in vitro motility experiments depicting Cin8-3GFP, rhodamine-labeled porcine brain-tubulin GMPCPP stabilized seeds, and yeast microtubules (denoted by arrowheads) visualized by interference reflection microscopy (IRM). Scale bars = 10 µm. (C) Quantification of Cin8 minus-end velocity. TUB2: n = 75 events; tub2-Δ438Q: n = 115 events, P = 0.051; tub2-Δ438: n = 116 events, P = 0.13. Statistics are Student’s t test of replicate medians compared to wild-type TUB2. Error bars mean ± 95% CI. (D) Quantification of Cin8-3GFP fluorescence intensity per microtubule (MT) length. TUB2: n = 180 microtubules; tub2-Δ438Q: n = 195 microtubules, P = 0.012; tub2-Δ438: n = 196 microtubules, P = 0.28. Statistics are Student’s t test of replicate medians compared to wild-type TUB2. Error bars mean ± 95% CI. * indicates P < 0.05. (E) Percentage of plus-end-directed motility events out of all events (plus-end directed, minus-end directed, or diffusive). Each point is a technical replicate. (F) Percentage of plus-end processive motility time out of all time spent in processive motility (plus-end- or minus-end-directed motility). Each point is a technical replicate.