Fig. 4. ZNF384 modulates Ku70/Ku80 dynamics at DNA damage sites.

a DNA pull-downs of biotinylated DNA with a 3’-overhang in the presence of His-Ku70/Ku80, His-MBP-ZNF384, or His-MBP alone or His-Ku70/Ku80 in combination with His-MBP-ZNF384 or His-MBP. Blots were probed for MBP and Ku80. Ku80 pull-down signals were normalized to that in the pull-down lacking His-MBP-ZNF384, which was set to 1. The mean from four independent experiments is indicated below the blot. His-MBP was not detectable in pull-downs. A representative experiment is shown. b GFP-Ku70 recruitment to 800 nm multiphoton tracks in RPE1-hTERT cells transfected with the indicated siRNAs (left panel). White triangles indicate irradiated regions. Quantification of the data is presented as the mean ± SD from >60 cells acquired in two independent experiments. Scale bar 5 μm. c Western blot analysis of ZNF384 expression in cells from (b). Tubulin is a loading control. d Representative images of RPE1-hTERT cells transfected with the indicated siRNAs, in which FRAP measurements were performed to assess the local turnover of GFP-Ku70 at the sites of DNA damage. DNA damage was induced in the region indicated with a dashed line. Subsequent FRAP was induced in a subarea within the DNA damage region, as indicated with an unbroken line. Images are pseudocolored according to the look-up table displayed on the right. Scale bar 4 μm. e Normalized FRAP curves from (d). f Association (k’_on) rates of GFP-Ku70 measured by FRAP after fitting of the curves from (e). g Dissociation (k_off) rates of GFP-Ku70 after fitting of the curves from (e). Data from (e–g) was collected from 15 cells per condition. Boxplot limits correspond to the 25th and 75th percentiles and the center line in the box indicates the median value from a representative of two independent replicates. Statistical significance was calculated using the unpaired Student’s t test, assuming unequal variances. Source data are provided as a Source Data file.