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. 2008 Oct 20;183(2):253–265. doi: 10.1083/jcb.200711150

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© 2008 Li et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 2. — The HINT1 protein is recruited to IRIF and associates with γ-H2AX. (A) Hint1 −/− MEFs were transiently transfected with FLAG-Hint1, grown on coverslips, and treated with 4 Gy of IR. Cells were then fixed after the indicated times and stained with an anti-FLAG antibody (green) together with an anti–γ-H2AX antibody (red) and counterstained for the nucleus with DAPI. Adjacent cells not expressing FLAG-Hint1 were used as an internal negative control. Repeat studies gave similar results. (B) The kinetics of HINT1 and γ-H2AX nuclear foci formation before and after IR treatment. For each genotype and time point, the number of foci per cell was analyzed in 20 images (error bars represent standard deviation). (C) Hint1 −/− MEFs were transiently transfected with FLAG-Hint1 or the control vector (p3xFLAG). 48 h later cells were treated with or without 4 Gy of IR, and whole cell lysates were prepared for immunoprecipitation using the FLAG antibody. Immunoblots were performed using either a FLAG antibody, a γ-H2AX antibody, or an ATM antibody. Repeat studies gave similar results. *, P < 0.05 compared with untreated cells.