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. 2019 Jun 19;47(14):7564–7579. doi: 10.1093/nar/gkz537

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — hTRAF6 promotes hDNA2 nuclear localization. (A) HeLa cells were transfected with an empty control vector (EV) or a vector encoding myc-hTRAF6. Endogenous hDNA2 protein in the CE and NE was analyzed by western blot analysis using an anti-hDNA2 antibody. GAPDH and histone H3 were used as CE and NE markers, respectively. (B) 3xFlag-hDNA2 was co-expressed with EV or a vector encoding WT or C70A (ubiquitin E3 ligase activity-deficient) mutant hTRAF6 in 293T cells. 3xFlag-hDNA2 in the CE and NE was analyzed by western blot analysis using an anti-Flag antibody. GAPDH and histone H3 were used as CE and NE markers, respectively. (C–E) 293T cells co-expressing 3xFlag-hDNA2 and myc-hTRAF6 were left untreated or were treated with (C) a small molecule inhibitor of TRAF6 E3 ligase activity (C25–140, 30 μM, 48 h), (D) a TRAF6/RANK interaction-disrupting peptide (T6DP, 100 μM, 24 h) or (E) a small molecule ATM kinase activity inhibitor (KU-55933, 10 μM, 16 h). 3xFlag-hDNA2 levels in the CE and NE were analyzed by western blot analysis using an anti-Flag antibody. GAPDH and histone H3 were used as CE and NE markers, respectively. (F) 293T cells expressing 3xFlag-hDNA2 were pre-treated with DMSO (control) or KU-55933 (10 μM, 16 h). The cells were left untreated or were treated with CPT (1 μM, 4 h). 3xFlag-hDNA2 levels in the CE and NE were analyzed by western blot analysis using an anti-Flag antibody. GAPDH and histone H3 were used as CE and NE markers, respectively.