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. 2017 Apr 15;31(8):816–829. doi: 10.1101/gad.297663.117

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© 2017 Natsume et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 1. — Construction of MCM2-mAID cells for artificial fork stalling. (A) A schematic representation of auxin-mediated proteolysis of MCM2-mAID. An auxin-dependent F-box protein, AFB2 of A. thaliana (AtAFB2), forms an E3 ubiquitin ligase with the endogenous SCF components. In the presence of auxin, MCM2-mAID is targeted by AtAFB2 for polyubiquitylation and subsequent destruction by the proteasome. (B) Evidence that clones 1 and 2 express the MCM2-mAID protein. (C) Time course of proteolysis of MCM2-mAID. Asynchronously growing MCM2-mAID cells were treated with auxin (indole-3-acetic acid [IAA]) before harvesting at the indicated time points. Ponceau staining shows a loading control. (D,E) MCM2-mAID cells were arrested in G1 phase and released into S phase after MCM2-mAID depletion. In control cells, DMSO replaced auxin. MCM2-mAID proteins were detected by immunoblotting using the anti-mAID antibody (D), and DNA content was measured by flow cytometry (E).