For significant differences Cohen’s d was calculated as a measure of effect size

at one of the his3 loci in VMYD16. Following sporulation and tetrad dissection, the required haploid sli15D::KanMX6 his3::sli15-20A::HIS3 Debio 1347 web strains were generated. Control strains expressing wild-type SLI15 were similarly generated using pRS303-SLI15. For creating the sli15-20D plasmid, a 1479 bp fragment of SLI15 encoding 20 substitutions to glutamate or aspartate was synthesized and subcloned as an NcoI-MfeI restriction fragment in place of the equivalent wild-type fragment in pRS303SLI15, generating pRS303-SLI15-20D. pRS303-SLI15-20D was integrated in VMYD16 as above. Following tetrad dissection, high levels of spore inviability were seen that were rescued following transformation with pCJ145. sli15D::KanMX6 his3::sli15-20D::HIS3 strains were therefore generated by sporulation and tetrad dissection of the pCJ145-containing strain, followed by two rounds of selection on 5-fluoroorotic acid to evict the plasmid. Spindle assembly checkpoint analysis Whole-cell extracts were made and immunoblotted as previously described. Briefly, for synchronization of cells in G1, 1.25 mg/ml a-factor was used. To prevent cells from entering the next cell cycle after release from a-factor arrest, a-factor was added back when small buds appeared in the majority of cells. Nocodazole was used at 30 mg/ml. pGAL-SCC1 shut-off was performed as previously described. Cell cycle progression was studied by monitoring budding and Pds1 levels in strains expressing PDS1-myc18. For lysing cells sequential NaOH and trichloroacetic acid treatment was used. Pds1-myc18 was detected by Western blotting with an anti-myc antibody. Cdc28 was detected using an anti-Cdc28 antibody from Santa Cruz Biotechnology. GFP was detected with anti-GFP. Sister CEN5 centromeres that remained unseparated at one end of the metaphase spindle were scored as mono-oriented, whereas sister CEN5 centromeres that showed dynamic separation and reassociation were scored as bioriented.Microcystin was used at 1 mM and recombinant human protein phosphatase 1 at 0.05 mg per assay. Phosphorylated proteins were separated by SDS-PAGE, detected by autoradiography and quantitated by liquid scintillation counting after excising the radiolabelled bands. Measurement of Sli15 and microtubule binding affinity using Biolayer Interferometry Tubulin purified from bovine brain was mixed with 20 mg biotin labeled tubulin in ice-cold PME buffer containing 1 mM GTP and 1 mM DTT was thawed and centrifuged to remove insoluble protein for 5 min at 42,000 rpm using a Beckman TLA100 rotor PubMed ID: at 4uC. To assemble microtubules, the reaction was incubated at 37uC for 30 min while taxol was added in a stepwise manner to a final concentration of 20 mM to stabilize microtubules. Microtubules were then centrifuged at 50,000 rpm for 5 min at 30uC and the pellet was resuspended in warm PME buffer containing 1 PubMed ID: mM GTP, 1 mM DTT and 20 mM taxol to a final concentration of 5 mM polymerized tubulin. Binding assays were carried out by biolayer interferometry at 25uC in solid black 96-well plates using an Octet Red384. Streptavidin-coated biosensors were equilibrated in Buffer A, then loaded with 5 mM biotinylated polymerized tubulin. Unbound MTs were removed in a 5 min wash with Buffer A, followed by a subsequent wash with Buffer A containing 10 mM biocytin to block free streptavidin sites. Biocytin was removed in a 5 min wash step with Buffer A and the system was equilibrated for 5 min with Buffer B glycerol, 5 mM b-mercaptoethanol, 100 mM im

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