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. 2014 Dec 23;34(3):326–343. doi: 10.15252/embj.201489184

Figure 8.

Figure 8

USP45 is recruited to sites of damage following UV-C irradiation
  1. U2OS cells were transiently transfected with the GFP–USP45 or GFP alone. Thirty-six hours post-transfection cells were irradiated with UV-C (150 J/m2) through a micropore filter (lower panels) or left untreated (upper panels). Five minutes post-irradiation, cells were fixed and co-immunostained with antibodies recognising cyclobutane pyrimidine dimer (CPD) DNA lesions and GFP. The white arrows indicate co-localisation between GFP–USP45 and CPD. Scale bar, 10 μm.
  2. U2OS wild-type (WT) and USP45 knockout (KO) cells were irradiated with UV-C (20 J/m2). At the indicated post-irradiation times, genomic DNA was extracted and subjected to Southern dot blot analysis using antibodies recognising CPD and dsDNA total antibody. Data are shown from a triplicate experiment in which each blot is derived from independent cells.
  3. Same as (B) but using a High Sensitivity CPD/Cyclobutane Pyrimidine Dimer Elisa kit (NM-MA-K001) from Cosmo Bio. For the DNA damage detection, the manufacturer protocol was followed. Absorbance at 492 was measured that represent amount of CPDs in each sample at indicated times. Calf thymus DNA, UV-C irradiated (10 J/m2) and not irradiated was used as positive and negative samples, respectively.