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. 2014 Jun 19;5(6):e1296. doi: 10.1038/cddis.2014.247

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Copyright © 2014 Macmillan Publishers Limited

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

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Constitutive overexpression of SIRT1 does not protect differentiated SH-SY5Y neuroblastoma cells from G93A-SOD1 toxicity. (a) Immunofluorescence analysis on SH-SY5Y cells untransfected or stably transfected for SIRT1 overexpression using antibodies against SIRT1. Scale bar: 10 μm. (b) Western blot analysis of 20 μg of total protein extract from cells either untransfected (Ctrl) or stably transfected for SIRT1 overexpression (SIRT1) using antibodies against SIRT1 and β-actin as a loading control. One representative blot from three independent experiments giving comparable results is shown. (c) SIRT1 activity was measured by a fluorometric assay on 40 μg of total protein extract from either untransfected (Ctrl) or stably transfected cells (SIRT1). Activity is reported as percent of control cells and as the mean±S.D. of three independent experiments with each sample done in triplicate. Values significantly different from relative controls are indicated with **P<0.01 with respect to Ctrl. (d) Cell viability of untransfected (Ctrl) and stably transfected (SIRT1) cells infected with adenoviral vectors coding for Wt-SOD1 (Wt) and G93A-SOD1 (G93A). Values are the mean±S.D. of three independent experiments in triplicate. Values that do not differ significantly are marked with n.s. (e) Total protein extracts from cells as in (d) and treated with 3 μM Ex527 were assayed after 48 h for Caspase-3 activity. Data are reported as the mean±S.D. of three independent experiments. Values that do not differ significantly with respect to the relative control are marked with n.s; ^#P<0.05 significantly different with respect to DMSO. (f) Western blot analysis of cleaved PARP in 20 μg of total protein extract from cells as in (d) and treated with DMSO (−) or 3 μM Ex527 (+). Antibodies against β- actin were used as loading control. One representative blot is shown from three independent experiments giving comparable results. (g) Densitometric analysis of n=4 experiments as in (f). n.s. indicates values that do not differ significantly