Skip to main content
. 2006 Jun 29;25(14):3286–3297. doi: 10.1038/sj.emboj.7601212

Figure 1.

Figure 1

DNA damage induces ubiquitination and degradation of Daxx in PML-NBs. (A) Inhibition of HA-Daxx degradation induced by MMS or UV by the proteasome inhibitor LLnL. HeLa cells transfected with pCMV-HA-Daxx were incubated with dimethylsulfoxide or LLnL (100 μM) before treatment with MMS or UV. Cell lysates were prepared at the indicated time points post-treatment and immunoblotted with anti-HA or anti-GAPDH (loading control) antibodies. (B) Degradation of endogenous Daxx in response to MMS or UV. HeLa cells were treated with MMS or UV and lysates prepared at the indicated time points post-treatment were immunoblotted with anti-Daxx antibody. (C) Localization of Daxx in PML-NBs. Untreated HeLa cells were immunostained with anti-Daxx and anti-PML antibodies. All images were obtained by confocal microscopy. Scale bar, 10 μm. (D) Degradation of endogenous Daxx in PML-NBs in response to MMS or UV. Untreated, MMS- or UV-treated HeLa cells were immunostained with anti-Daxx antibody at 3.5 h post-treatment. Scale bar, 10 μm. (E) DNA damage-induced ubiquitination of Daxx. HeLa cells were cotransfected with pCMV-HA-Daxx and pCMV-Flag-ubiquitin, and either left untreated or treated with UV or MMS. HA-Daxx was immunoprecipitated (IP) from lysates of these cells using anti-HA-beads and Daxx ubiquitination was detected by immunoblotting (IB) with anti-ubiquitin antibody. (F) Degradation of full-length Daxx protein only. HeLa cells were transfected with pCMV-HA-Daxx full-length or the deletion mutants HA-Daxx1–501 or HA-DaxxC501. Transfected cells were left untreated or treated with MMS or UV and lysed at the indicated time points post-treatment. Cell lysates were immunoprecipitated with anti-HA beads and immunoblotted with anti-HA antibody.