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. 2017 Jul 28;8(40):68493–68516. doi: 10.18632/oncotarget.19663

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Copyright: © 2017 Kollipara et al.

This article is distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use and redistribution provided that the original author and source are credited.

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(A) Targeted transcript studies of ATXN10 utilizing LCs derived from healthy controls and MSS-patients (same batch of cells used for the proteomic studies) revealed a 1.6-fold increase in patient-derived LCs. (B) Immunoblot-based examination of ATNX10 level in sera derived from woozy animals and wildtype littermates (as well as one heterozygous animal). Whereas in sera of the wildtype littermates and in the heterozygous animal a band at 37 kDa could consistantly be detected, in woozy sera the utilized antibody detected a band with a molecular weight lower than 37 kDa. Coomasie blue staining has been carried out to demonstrate equal protein loading. Further immunoblot-based investigation of ataxin-10 protein level in whole cerebellar protein lysates derived from 6- and 26-week old woozy and control animals shows a reduced netto abundance in 6-week old animals when PC degeneration in initiated (lower left panel). In contrast, in whole cerebellar protein lysates derived from 26-week old animals (surviving PC population in the vestibulocerebellum), increased abundance can be detected (lower panel in the middle). In both, the 6- and the 26-week old animals an addional band (of higher molecular weight; approx. 65 kDa) showing similar regulation of protein abundance can be detected. Coomasie blue staining has been carried out to demonstrate equal protein loading. Investigation of whole quadriceps muscle protein lysates derived from 26-week old woozy and control animals revealed increased ataxin-10 abundance in the diseased muscle of the Sil1 mutant animals (lower right panel). Here, only one band at the predicted ataxin-10 size of 53 kDa could be detected. Coomasie blue staining has been carried out to demonstrate equal protein loading. (C) Immunohistochemistry of paraffin sections of paraformaldehyde-fixed specimens of 6-week and 26-week old control (wt) and of Sil1 mutant mice (woozy) revealed reduced ataxin-10 abundance in the vulnerable neocerebellum at the age of 6- and 26-week old woozy animals compared to the wildtype littermates (C1-C4) and increased ataxin-10 abundance in the non-vulnerable vestibulocerebellum at the age of 6- and 26-week old woozy animals compared to the wildtype littermates (C5-C8). Increased immunoreactivity can be observed in PCs and the neuropil. Sclale bars = 40 μm. No obvious changes in ataxin-10 abundance can be detected in the spleen, the heart and the kidney of Sil1 mutant animals compared to the wildtype controls (C9-C14; 26-week old). Scale bars: in C9 and C10 = 50 μm, in C11 and C12 = 60 μm, in C13 and C14 = 40 μm. Increased immunoreactivity in motoneurons of Sil1 mutant animals compared to the wildtype controls (C15 and C16; 26-week old). Scale bars = 50 μm. Increased ataxin-10 immunoreactivity can be observed in diseased and degenerating muscle fibres of Sil1 mutant animals compared to the wildtype controls (C17-C21; 26-week old). Scale bars: in C17 and C18 = 65 μm, in C19 = 4 μm, in C20 = 30 μm, in C21 = 100 μm.

(A) Targeted transcript studies of ATXN10 utilizing LCs derived from healthy controls and MSS-patients (same batch of cells used for the proteomic studies) revealed a 1.6-fold increase in patient-derived LCs. (B) Immunoblot-based examination of ATNX10 level in sera derived from woozy animals and wildtype littermates (as well as one heterozygous animal). Whereas in sera of the wildtype littermates and in the heterozygous animal a band at 37 kDa could consistantly be detected, in woozy sera the utilized antibody detected a band with a molecular weight lower than 37 kDa. Coomasie blue staining has been carried out to demonstrate equal protein loading. Further immunoblot-based investigation of ataxin-10 protein level in whole cerebellar protein lysates derived from 6- and 26-week old woozy and control animals shows a reduced netto abundance in 6-week old animals when PC degeneration in initiated (lower left panel). In contrast, in whole cerebellar protein lysates derived from 26-week old animals (surviving PC population in the vestibulocerebellum), increased abundance can be detected (lower panel in the middle). In both, the 6- and the 26-week old animals an addional band (of higher molecular weight; approx. 65 kDa) showing similar regulation of protein abundance can be detected. Coomasie blue staining has been carried out to demonstrate equal protein loading. Investigation of whole quadriceps muscle protein lysates derived from 26-week old woozy and control animals revealed increased ataxin-10 abundance in the diseased muscle of the Sil1 mutant animals (lower right panel). Here, only one band at the predicted ataxin-10 size of 53 kDa could be detected. Coomasie blue staining has been carried out to demonstrate equal protein loading. (C) Immunohistochemistry of paraffin sections of paraformaldehyde-fixed specimens of 6-week and 26-week old control (wt) and of Sil1 mutant mice (woozy) revealed reduced ataxin-10 abundance in the vulnerable neocerebellum at the age of 6- and 26-week old woozy animals compared to the wildtype littermates (C1-C4) and increased ataxin-10 abundance in the non-vulnerable vestibulocerebellum at the age of 6- and 26-week old woozy animals compared to the wildtype littermates (C5-C8). Increased immunoreactivity can be observed in PCs and the neuropil. Sclale bars = 40 μm. No obvious changes in ataxin-10 abundance can be detected in the spleen, the heart and the kidney of Sil1 mutant animals compared to the wildtype controls (C9-C14; 26-week old). Scale bars: in C9 and C10 = 50 μm, in C11 and C12 = 60 μm, in C13 and C14 = 40 μm. Increased immunoreactivity in motoneurons of Sil1 mutant animals compared to the wildtype controls (C15 and C16; 26-week old). Scale bars = 50 μm. Increased ataxin-10 immunoreactivity can be observed in diseased and degenerating muscle fibres of Sil1 mutant animals compared to the wildtype controls (C17-C21; 26-week old). Scale bars: in C17 and C18 = 65 μm, in C19 = 4 μm, in C20 = 30 μm, in C21 = 100 μm.