Figure 6.

USP39 is a key factor in creating NHEJ complexes by interplay with PAXX, APTX, XRCC4 and LIG4. (A) GST-free USP39 directly interacts with recombinant GST-APTX, GST-XRCC4 and GST-LIG4, but not GST-PAXX. Indicated recombinant proteins were purified from insect cells and used in a USP39 overlay assay. GST was used as negative control. (B and C) Cell extracts from HEK293FT-expressing FLAG-tagged NHEJ factors (B) or FLAG-USP39 (C) were immunoprecipitated with anti-FLAG antibody and then analyzed by immunoblotting. (D) Endogenous USP39 interacts more strongly with endogenous LIG4 in the DNA-damaged state. The cell lysates derived from normal or DNA-damaged cells were immunoprecipitated with anti-USP39 antibody, and then analyzed for interaction between endogenous USP39 and endogenous LIG4. (E–G) Depletion of USP39 leads to failure of proper accumulation of endogenous LIG4 at DSBs. siRNA-targeting USP39 was transfected into U2OS cells and then stripe formation of endogenous LIG4 was analyzed. Yellow arrows indicate the area of signal intensity measurement (E). The accumulated level of γH2AX or LIG4 at the DNA lesions was analyzed by Nikon NIS software (F). Relative intensity of endogenous LIG4 accumulated at DSBs was monitored in cells expressing either siCtrl or siUSP39. n indicates the total cell number used in quantification (G). Statistical significance was determined by the Student's t-test. Scale bars, 5 μm. Data represent the mean ± s.e.m. of three independent experiments. ***P ≤ 0.001.