Skip to main content
. 2016 May 4;17(6):874–886. doi: 10.15252/embr.201541685

Figure EV2. Co‐immunofluorescence experiments showing co‐localization of HUWE1 and PCNA .

Figure EV2

  • A–C
    Representative immunofluorescence micrographs of U2OS cells untreated (A), treated with 2 mM HU for 24 h (B), or exposed to 40 J/m2 UV and analyzed 2 h later (C).
  • D
    Quantifications showing the percentage of PCNA foci co‐localizing with HUWE1 foci and, reciprocally, the percentage of HUWE1 foci co‐localizing with PCNA foci. Only cells with more than five foci were analyzed. The average of three experiments, with standard errors, is shown. In total, 191 HUWE1 and 156 PCNA foci were counted.
  • E
    HUWE1‐knockout cells corrected with wild‐type or PIP mutant were subjected to immunofluorescence using HUWE1 and PCNA antibodies. Similar to the endogenous protein, Myc‐HUWE1 wild‐type forms UV‐ and HU‐induced chromatin foci. In contrast, the PIP‐box mutant does not co‐localize with PCNA after damage exposure. Shown are representative micrographs, as well as quantifications of the percentage of PCNA foci co‐localizing with HUWE1 and, reciprocally, the percentage of HUWE1 foci co‐localizing with PCNA, in wild‐type and PIP mutant‐corrected cells. Only cells with more than five foci were analyzed. The average of three experiments, with standard errors, is shown. A total number of foci counted were as follows: for the HU experiment: 162 HUWE1‐WT foci with 144 PCNA foci; 196 HUWE1‐FF foci with 163 PCNA foci. For the UV experiment: 146 HUWE1‐WT foci with 121 PCNA foci; 101 HUWE1‐FF foci with 78 PCNA foci.