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. 2012 Sep 16;40(21):10795–10808. doi: 10.1093/nar/gks850

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© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — Requirement of the UBZ and PIP domains of Spartan and Rad18 for Spartan localization to replication forks. (A) The UBZ and PIP domains of Spartan are essential for foci formation and colocalization of Spartan with PCNA. HeLa cells transiently transfected with plasmids expressing WT, UBZ, PIP, PIP-UBZ or SprT domain mutant FLAG-Spartan proteins were processed for indirect immunofluorescence with antibodies against FLAG and PCNA. (B) The localization of Spartan depends on Rad18. Knock out HCT116 RAD18-/- and RAD18+/+ cells were transfected with plasmids expressing GFP-Spartan of which localization was compared in the two cell lines (upper panel). Localization of GFP-Spartan in RAD18-/- cells and DsRed-Rad18 expressing RAD18-/- cells (lower panel) was also compared. The percentages of GFP-Spartan expressing cells that display more than five Spartan foci were determined from three independent experiments and standard deviation was also calculated (right panel). (C) Spartan associates with mono- and polyubiquitin PCNA in vivo. HEK 293 cells were transfected with various combinations of control or USP1 shRNAs, HA-PCNA and FLAG-Spartan. Cell extracts were subjected to immunoprecipitation with anti-FLAG antibody, and the coimmunoprecipitated unmodified-, mono- and polyubiquitinated PCNAs were detected by western blotting using anti-HA antibody.