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. 2010 May 11;19(15):2974–2986. doi: 10.1093/hmg/ddq202

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© The Author 2010. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — MutSOD1s induce a conformational change in Bcl-2 leading to exposure of the toxic BH3 domain in cells, mouse and human spinal cords. (A) SH-SY5Y cells, which express endogenous Bcl-2, were transfected with SOD1 (WT, G37R or G93A) and Bcl-2 conformation assessed by immunoprecipitation with the α-Bcl-2/BH3 and α-Bcl-2/pocket antibody, respectively. Immunoprecipitated proteins were analyzed by WB with anti-Bcl-2 antibody against the N-terminal domain (left panel). Total amount of Bcl-2 was assessed by WB (left panel, bottom lane). In the presence of mutSOD1s (G37R and G93A), there is an increased exposure of the toxic BH3 domain paralleled by a decrease in the pocket region. The plot in the right panel shows the ratio BH3/pocket as analyzed by densitometric analysis of the immunoprecipitated Bcl-2 with the Quantity One software. (B) The α-Bcl-2/BH3 and α-Bcl-2/pocket antibodies were used to immunoprecipitate Bcl-2 from spinal cord homogenates of 130-day-old transgenic mice expressing either SOD1-WT or SOD1-G93A. In SOD1-G93A mice there is an increased exposure of the toxic BH3 domain of Bcl-2 compared with age-matched transgenic SOD1-WT mice, and this is paralleled by a decreased exposure of the pocket. (C) Conformational changes in Bcl-2 were then assessed over time in the SOD1-G93A mice. Exposure of the toxic BH3 domain appears prior to and peaks at disease onset (left panel). Densitometry analysis of the ratio BH3/pocket is shown in the plot on the right. (D) Immunoprecipitation of post-mortem human SOD1-A4V spinal cord shows increased exposure of the BH3 domain (shown as *) paralleled by a decreased exposure of the pocket region in human ALS.