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. 2011 Jul 15;22(14):2448–2457. doi: 10.1091/mbc.E09-01-0029

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© 2011 Barnhart et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 5: — MAD1/mad1Δ MAD2/mad2Δ cells arrest in response to loss of tension and loss of attachment. (A) Western blots to detect the relative levels of Mad1-TAP (top) or Mad2-TAP (bottom) epitope-tagged strains that are homozygous diploids, heterozygous diploids, or double heterozygous diploids. Both Mad1 and Mad2 proteins levels appear to be lowered by approximately one-half the amount in the heterozygous diploids and double heterozygous diploids relative to the homozygous diploid in comparison to mixed ratios of homozygous TAP-tagged diploids and homozygous nulls (n = 3). (B) The indicated strains were tested for their ability to proliferate in the absence (CDC20-127 expressed; left) or presence (CDC20-127 repressed; right) of the spindle checkpoint. Serial fourfold dilutions of all strains were spotted onto plates containing 0 μg/ml doxycycline (left) or 10 μg/ml doxycycline (right) and grown for 2 d. (C) MAD1/mad1Δ MAD2/mad2Δ double heterozygotes have a mitotic delay in response to loss of tension on chromosomes. Error bars represent the SD of three separate trials. (D) MAD1/mad1Δ MAD2/mad2Δ double heterozygotes do not arrest in response to a loss of tension on all mitotic chromosomes. We measured cell cycle progression by Western blotting after release from a G1 arrest in the absence of the MCD1. Western blots against Myc (top) or actin (bottom) were performed (n = 3). In the absence of sister chromatid cohesion, wild-type and MAD1/mad1Δ cells display an extended arrest. MAD1/mad1Δ MAD2/mad2Δ diploid cells do not arrest in response to a loss of cohesion on all mitotic chromosomes.