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. 2018 Feb 1;29(3):339–355. doi: 10.1091/mbc.E17-08-0511

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© 2018 Robison et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 5: — smc3-D667p has reduced acetylation at K113. (A) Regimen used to assess Smc3-K113 acetylation in mid–M phase–arrested cells. Early log phase cultures were treated with 0.75 mM auxin for 1 h to deplete Smc3-3V5-AIDp, and then nocodazole was added, and cultures were incubated 3 h to arrest cells in mid–M phase. (B) Reduced K113 acetylation of smc3-D667p. Haploid ECO1-AID (VG3633-2D), SMC3-AID (VG3651-3D), SMC3 SMC3-AID (BRY474), and smc3-D667 SMC3-AID (BRY482) cultures grown as described in A. Protein extracts were made and subjected to SDS–PAGE and then analyzed by Western blot. Antibodies against Smc3-K113 acetylation (Smc3-ac) are shown as short and long exposures; anti-Mcd1p antibodies (Mcd1p) serve as control for cohesin levels and antibodies against tubulin (Tub1) for a loading control. (C) Regimen used to determine the kinetics of Smc3-K113 acetylation establishment within a single cell cycle. Log phase cultures grown in YPD at 23°C were arrested in G1 using alpha factor, treated with auxin to deplete Smc3p-AID in G1, and then released into fresh YPD containing auxin and nocodazole to synchronously arrest cells in mid–M phase (Materials and Methods). (D) smc3-6HA-D667p has reduced acetylation in S phase but acetylation remains in mid–M phase. Haploid SMC3-AID cells expressing Smc3-6HAp (BRY604, left) or smc3-6HA-D667p (BRY602, right) were grown as described in C. Aliquots were taken at the indicated time points, and protein extracts were made. A small portion was reserved for total protein, and then anti-HA antibody was added to immunoprecipitate Smc3-6HAp or smc3-6HA-D667p (Materials and Methods). Samples were subjected to SDS–PAGE and then analyzed by Western blot. Antibodies against Smc3-K113 acetylation (Smc3-ac) and both short and long exposures are shown for better comparison. Antibodies were used to monitor levels of the Smc3p (HA) and Mcd1p (Mcd1) cohesin subunits and anti-Tubulin antibodies (Tub1) used as a loading control. Samples were also collected to assess DNA content by flow cytometry (right side). (E) Similar levels of K113 acetylation in smc3-D667 and eco1-203 at permissive temperature. Haploid strains SMC3-AID (VG3651-3D), SMC3 SMC3-AID (BRY474), smc3-D667 SMC3-AID (BRY482), and eco1-203 (VG3506-5D) were treated as described in A. Protein extracts were made and then subjected to SDS–PAGE and Western blot analysis. Antibodies against Smc3-K113 acetylation (Smc3-ac) and both short and long exposures are shown for better comparison. Anti-MCD1 antibodies (Mcd1) were used as a control for cohesin levels and anti-Tubulin antibodies (Tub1) for a loading control.