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. 2010 Nov 8;107(47):20364–20369. doi: 10.1073/pnas.1011069107

Fig. 3.

Fig. 3.

Eco2 is required for recruitment of sororin to chromatin. (A) Nuclei were added to interphase extracts that were mock depleted (mock) or immunodepleted of Eco1 (ΔEco1), Eco2 (ΔEco2), Eco1 and Eco2 together (ΔEco1/2), cohesin (ΔSmc3), or sororin (ΔSor). At the indicated times, chromatin fractions were isolated and analyzed by immunoblot for the presence of topoisomerase (loading control), Smc3, Eco2, and sororin. (B) Protein levels in total extract. Depleted extracts used in A were analyzed by immunoblot for topoisomerase II, Smc3, or Eco2 (Eco1 was undetectable in this experiment, and sororin was obscured by background bands in total extract). (C) Rescue of sororin recruitment with recombinant Eco2 interphase extract was depleted of Eco2 and either left unsupplemented (−) or supplemented with recombinant 6His–Eco2 at 0, 60, or 120′ as indicated (Upper). Chromatin-bound fractions were collected at both 90 and 150′ after sperm addition and analyzed by immunoblot for the presence of Smc3, Eco2, and sororin. Also shown are immunoblots for Eco2 and Smc3 in total extracts (extract) (Upper). Coomassie-stained histones were used as loading controls. (D) Eco2 recruitment to chromatin independent of replication interphase extracts were supplemented with geminin, p27, or aphidicolin as in Fig. 1. Chromatin-associated proteins were analyzed at the indicated times after the addition of sperm nuclei.