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Fig. 6. (A) Centromeric cohesion loses its elasticity in the absence of Slk19. cdc20 ${Delta}$ GAL-CDC20 (US3444) and cdc20 slk19 ${Delta}$ GAL-CDC20 (US3448) strains carrying TetO/GFP-TetR constructs were arrested in G1 using ${alpha}$ factor and then released into YEPD for 240 minutes to arrest them in G2-M. Each culture was divided into two halves; nocodazole was added to one half of the culture for 1.5 hours. Samples were collected and proportion of cells (a total of 150 cells were counted) with divided centromeric markers was determined (bottom left panel; also see Materials and Methods). The distance between the centromeric markers is graphically depicted in the bottom right panel. (B) Physical association of Slk19 and Scc1. (a) WT cells with endogenously tagged SLK19-HA₆ and SCC1-cmyc₁₈ at their respective loci (US3779) were arrested in G1 ( ${alpha}$ factor), S phase (hydroxyurea) and metaphase (nocodazole). Immunoprecipitates from the cell extracts were obtained by using rabbit anti-HA or rabbit anti-cmyc beads and analyzed by western blotting using mouse anti-cmyc or mouse anti-HA antibodies respectively. ^*,** Indicate the cleaved forms of Scc1 and Slk19, respectively. The extracts were analyzed by western blotting. (b) Negative control. Extracts were immunoprecipitated with goat anti-HA or rabbit anti-cmyc antibodies and analyzed by western blots using mouse anti-cmyc or mouse anti-HA antibodies, respectively. Lane 1: WT with endogenously tagged SLK19-HA₆ and SCC1-cmyc₁₈ at their respective loci (US3779); Lane 2: untagged WT (US1363); Lane 3: WT with endogenously tagged SCC1-cmyc₁₈ and untagged Slk19 (US3335); Lane 4: WT with endogenously tagged SLK19-HA₆ and untagged Scc1 (US4305). (c) Extracts were immunoprecipitated with goat anti-HA or rabbit anti-cmyc beads and immunoprecipitates were analyzed by western blotting using mouse anti-cmyc or mouse anti-HA antibodies, respectively. Lane 1: Extracts from asynchronously growing untagged WT (US1363); Lane 2: WT carrying endogenously tagged SLK19-HA₆ and SCC1-cmyc₁₈ (US3779); Lane 3: WT with endogenously tagged SCC1-cmyc₁₂ and carrying slk19-( ${Delta}$ 1-77)-HA₃ on CEN plasmid (US3972). (d) The results from a MBP pull down assay. Bacterially produced MBP or MBP-Slk19 fusion proteins immobilized onto beads were incubated with various extracts and analyzed by western blotting against mouse anti-cmyc antibodies or rabbit anti-Clb2 antibodies. Lane 1: MBP-Slk19 beads with extracts containing SCC1-cmyc₁₈ (from US3335); Lane 2: MBP beads with extracts containing SCC1-cmyc₁₈ (from US3335); Lane 3: MBP-Slk19 beads; Lane 4: MBP beads; Lane 5: extracts containing SCC1-cmyc₁₈ (from US3335); Lane 6: extracts from untagged WT (US1363) as a negative control.

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