The Journal of Neuroscience, October 26, 2005, ():
Dysfunction of the Cholesterol Biosynthetic Pathway in Huntington's Disease
J. Neurosci. Valenza et al.
25: 9932
Supplemantal data
Files in this Data Supplement:
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Supplemental material.
- supplemental material
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Supplementary Figure 1 online Legend:
Hydroxy-methyl-glutaryl CoA reductase (HMGCoAred), cytocrome P450 lanosterol 14-alpha-demethylase (Cyp51) and 7-dehydroxycholesterol reductase (7dhcred) gene transcription was reduced also in HD brain from patients with advanced grades (III and IV). The figure shows the semiquantitative radioactive RT-PCR analysis of human post-mortem cortex from grade III (HD III) and from grade IV (HD IV) versus control (Co) and, in the graph, the quantitative analysis of HMGCoAred, Cyp51 and 7dhcred mRNA levels. The peak densitometric area was normalised over the peak densitometric area of the ?-actin band. Tha data are expressed as percentage of control value
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Supplementary Figure 2 online Legend:
Total cholesterol content in striatum (left panel) and cortex (right panel) from 10-week-old R6/2 transgenic mice. The cholesterol levels in both regions were lower in the HD mice (R6/2, white columns) than in the control mice (co, grey columns). The data were normalized on the basis of tissue weight and protein content. The values for striatum (co, n = 5; HD, n = 5) and cortex (co, n = 9; HD, n = 9) are expressed as percentages of controls. ** P <0.01 vs control tissues (co) (ANOVA).
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Supplementary Figure 3 online Legend:
Western blot analyses of total lysates of parental (P) brain cells, and brain cells expressing wild-type (wt) or mutant huntingtin (mu) (panel a), and from control (co) and 10-week-old R6/2 brains (panel b). The immunoreactive band corresponding to SREBP1 (125 kDa) was equally expressed in the HD and mouse cells vs their respective controls.
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Supplementary Figure 4 online Legend:
Western blot analyses of nuclear extracts showing that nuclear SREBP2 levels were lower in inducible mutant huntingtin-expressing cells (HD43) than in CHO cells (positive control). The graph shows the quantitative densitometric evaluations of the nuclear form of SREBP2 in comparison with histone H1, expressed in percentages.
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Supplementary Figure 5 online Legend:
Western blot analyses of nuclear extracts of (a) inducible mutant htt cells (HD43), and (b) of cytoplasmic (left) and nuclear extracts (right) of R6/2 and control brain cells. In (a), R6/2 extracts were loaded as a control for aggregates and their recognition by the anti-EM48 antibody. In both (a) and (b), the whole gels (including the wells in the stacking) were transferred to nitrocellulose membranes. The membranes were blotted with anti-SREBP1 antibody (*) and then with the anti-EM48 antibody (#), which specifically recognises the aminoterminal portion of mutant huntingtin and its aggregates, in order to reveal any immunoreactive bands corresponding to SREBPs that coincide with the EM48 immunoreactive band. As previously shown [Sipione et al., 2002], there are no aggregates in our inducible mutant huntingtin-expressing cells but, as expected, the anti-EM48 antibody recognized mutant huntingtin aggregates in the R6/2 total (a), and cytoplasmic and nuclear extracts (b).
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Supplementary Figure 6 online Legend:
Western blot analyses of the electroporated primary cells using anti-huntingtin 1HU-4C8 (Euromedex) and anti-GPF antibodies (clones 7.1 and 13.1, Roche).
