Fig. 4.

GFI1 activities are independent of DNA damage. a GFI1-Flag fusion protein was immunoprecipitated in 293T cells treated with 5 Gy IR and allowed to recover for the indicated amount of time. Extracts were separated by SDS–PAGE and blotted for the indicated proteins. b SupT1 cells were spread on glass slides 15 min and 1 h after irradiation using a Cytospin, stained for endogenous Gfi1 and γ-H2AX and visualized for immunofluorescence by confocal microscopy. Control cells stained without primary antibody but with secondary antibodies are shown. c U2OS cells carrying a LacO array and expressing a LacR-Fok1-mCherry endonuclease were transfected with a vector expressing the GFI1-GFP fusion protein. These cells were plated on cover glass, stained for γ-H2AX and visualized for immunofluorescence by confocal microscopy. d U2OS cells expressing a GFI1-GFP fusion protein were exposed to 405 nm UV micro-irradiation and the recruitment of the GFI1-GFP fusion protein to the site of damage was quantified by confocal microscopy. Average signal intensity is shown with error bars representing s.d. Recruitment of Ku80-mRuby2 fusion protein and GFP protein are shown as controls. Representative images of selected time points are shown on the right. Scale bar represents 10 μm