Purified Vpr induces double-strand breaks in vitro.

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Purified Vpr induces double-strand breaks in vitro. Purified Vpr induces double-strand breaks in vitro. A, a scheme of the protocol used to detect Vpr-induced double-strand breaks in isolated nuclei. B, Vpr localization in isolated nuclei. Isolated nuclei from HT1080 after incubation with Vpr were immunostained by α-Vpr (mAb8D1) and the images were captured by a laser confocal microscopy. The Z-series of optical sections collected at 1 μm steps of the cells were presented. Vpr (red; middle), DNA staining by Hoechst (blue; top) and their merged images (bottom) are shown. Without Vpr incubation, any signals by α-Vpr immunostaining were not detected in isolated nuclei (data not shown). Bar, 10 μm. C, PFGE analysis of double-strand breaks in isolated nuclei treated with Vpr. Molecular mass markers (lane 1), control cells (lane 2), cells subjected to X-ray irradiation (lane 3), and isolated nuclei without (lane 4) or with 10 μmol/L Vpr (lane 5). Arrow, double-strand breaks. D, Ni-NTA pull-down assay with His6-tagged Vpr on isolated nuclei. Precipitated proteins bound to His6-tagged Vpr (lane 2) and the control precipitates (lane 1) are indicated. *, His6-Vpr–specific bands. Hiroaki Tachiwana et al. Cancer Res 2006;66:627-631 ©2006 by American Association for Cancer Research