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. 2019 Jun 21;8:e44736. doi: 10.7554/eLife.44736

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© 2019, Srinivasan et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 6. — (A) CDK1 inhibition leads to cohesin cleavage: cdc28-as1(K25423) cells were arrested in G2 with nocadazole and treated with 5 μM 1NMPP1. Samples were drawn at indicated times and subjected to western blot analysis with the indicated antibodies. (B) Cohesin expressed at high CDK1 levels is resistant to Wapl-independent release: scc3K404E cdc28-as1 (K25437) and scc3K404E scc2-45 cdc28-as1 (K25440) containing 2C Smc1 and 2C Smc3 along with galactose inducible 2C Scc1^NCwere arrested in G2 with nocadazole. Scc1^NC expression induced by galactose addition for 60 min. Glucose and 1NMPP1 were added to the cultures for 60 min followed by temperature shift to 37°C for 20 min. Samples drawn before (0) and after temperature shift (20) were analysed by mini-chromosome IP following in vivo cohesin crosslinking with BMOE. (C) CDK1 is required to abolish Wapl-independent release: Strains described in (B) were arrested in G2. Followed by 1NMPP1 addition for 60 min. After this, galactose was added to the cultures to induce 2C Scc1^NC for 60 min. Glucose was added to the cultures followed by temperature shift to 37°C for 20 min. Samples drawn before (0) and after temperature shift (20) were analysed by mini-chromosome IP following in vivo cohesin crosslinking with BMOE.