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. 2014 Apr 15;25(8):1202–1215. doi: 10.1091/mbc.E13-07-0430

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© 2014 Datta et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 5: — Oxidative stress and progerin inhibit Ubc9 import by a mechanism that is independent of Ubc9 cysteine 93 oxidation. (A) Localization of Flag-Ubc9 (WT and C93S mutant; green) in HeLa cells treated with 200 μM H₂O₂ for 10 min. (B) Localization of Flag-Ubc9 (WT and C93S mutant; green) in HeLa cells cotransfected with HA-lamin A or HA-progerin (red). Scale bars, 10 μm. (C, D) Histograms showing N/C values (bin size, 0.5) of Flag-Ubc9 and Flag-Ubc9-C93S in cells expressing HA-lamin A (black) and HA-progerin (red). (E) Immunoblotting for the Ubc9-Uba2 disulfide in control (8469) and HGPS (1972, 1498, and 3199) fibroblasts. The electrophoretic position of the Ubc9-Uba2 disulfide (arrow) and a nonspecific cross-reaction (asterisk) are indicated. Note that the Ubc9 C93S mutant was expressed at a low level in these experiments to avoid dominant-negative effects on nuclear SUMOylation sufficient to disrupt the Ran gradient (Kelley et al., 2011).