Figure 3.

Plasmid–cohesin association in metaphase cells expressing two differentially tagged cohesin moieties. (A) The types of sister plasmid cohesion expected from the embrace, hand-cuff and bracelet models in presence of cohesin(Mcd1-HA6) and cohesin(Mcd1-Myc13) in equivalent amounts are indicated (see also Table 1). Whereas Mcd1-HA6 in cohesin could be cleaved by TEV protease, Mcd1-Myc13 could not. (B) Aliquots of cell lysates were probed by western analysis using HA- or Myc-antibody to reveal relative levels of cohesin(Mcd1-HA6) or cohesin(Mcd1-Myc13). Data are shown for the haploid strains expressing either cohesin(Mcd1-HA6) (lane 1) or cohesin(Mcd1-Myc13) (lane 2) and the diploid generated from them expressing both Mcd1 variants (lanes 3–7). Signals from the two antibodies were normalized using aliquots of ∼75% pure Cre recombinase tagged at its carboxyl-terminus with HA6 as well as Myc13. The mean Myc13 to HA6 signal intensity was 1.83 ± 0.18. The dilution factor between Cre samples stained by Coomassie blue (right) and the corresponding ones analyzed by western blotting (left) was 500 to 1. (C) Aliquots of cell lysates from the haploid and diploid strains, run as in B, were probed using an antibody to native Mcd1. The mean ratio of Mcd1-Myc13 to Mcd1-HA6 was 0.96 ± 0.11. (D) pSG4 molecules from the cleared lysate were first immobilized on IgG beads, and then released from them by disrupting TetO–TetR interaction using anhydrotetracycline. (E and F) Following treatment with EcoRI or TEV protease, plasmid pull-down was attempted using HA- or Myc-antibody.