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. 2014 Dec 10;43(1):282–294. doi: 10.1093/nar/gku1301

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — eGFP-Polκ foci formation after MMS treatment. (A) Detection of PCNA-Ub after MMS treatment by immunoblotting. Chromatin-bound proteins were isolated from WT or Pcna^K164R cells (MEFs) that were mock treated or continuously exposed to MMS (0.75 mM) for 6 h and separated with SDS-PAGE. After blotting, the membrane was cut below the 38 kDa marker and subsequent antibody staining and ECL was performed separately. The part of the membrane containing proteins with molecular weights >38 kDa (containing PCNA-Ub) was exposed for the maximum time, while the lower part of the membrane (<38 kDa proteins, containing unconjugated PCNA) was exposed only shortly. The asterisk indicates PCNA modified with two ubiquitin moieties. A representative experiment is shown. (B) WT and Pcna^K164R MEFs containing eGFP-Polκ were continuously exposed to MMS (0.75 mM) for 6 h after which they were fixed in 4% paraformaldehyde and analyzed with fluorescent microscopy. The average of three independent experiments is shown ±SD.