Fig. 5: The C2H2 motifs and N-terminus of ZNF384 are required for Ku70/Ku80 loading at DSBs.
From: Zinc finger protein ZNF384 is an adaptor of Ku to DNA during classical non-homologous end-joining
a Pull-downs of the indicated GFP fusion proteins in U2OS Flp-In/T-Rex cells. Blots were probed for Ku70, Ku80, Tubulin, and GFP. The data shown represent three independent experiments. b In vitro Ku80 pull-down in the presence or absence of His-Ku70/Ku80 and His-MBP, His-MBP-ZNF384, or His-MBP-∆N-terminus. Control IP contained beads only. Blots were probed for Ku80 and MBP. The data shown represent two independent experiments. c Western blot analysis of the expression of endogenous ZNF384 and ectopic GFP-ZNF384 full-length and deletion mutants. Tubulin is a loading control. The asterisk (*) indicates endogenous ZNF384. The data shown represent three independent experiments. d Accumulation of endogenous Ku80 at 365 nm UV-A tracks in BrdU-sensitized U2OS Flp-In/T-Rex cells expressing siRNA-resistant doxycycline (dox)-inducible GFP-ZNF384, GFP-ZNF384 ΔC2H2, and GFP-ZNF384 ΔN-terminus following transfection with the indicated siRNAs. Cells were subjected to laser micro-irradiation and 10 min later fixed and immunostained. White triangles indicate irradiated regions (upper panel). Quantification of endogenous Ku80 levels in laser tracks is presented as ±SEM of >150 cells acquired in 3 independent experiments (lower panel). e As in (d), except for PAR (upper panel). Quantification of endogenous PAR levels in laser tracks is presented as the mean ± SEM from three independent experiments. Data were normalized to siLuc, which was set to 100% (lower panel). Statistical significance was calculated using the unpaired Student’s t test, assuming unequal variances. Scale bar 5 μm. Source data are provided as a Source Data file.
