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Figure 2 Greatly reduced TDP2 protein and activity in 850-BR patient fibroblasts Greatly reduced TDP2 protein and activity in 850-BR patient fibroblasts.

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Presentation on theme: "Figure 2 Greatly reduced TDP2 protein and activity in 850-BR patient fibroblasts Greatly reduced TDP2 protein and activity in 850-BR patient fibroblasts."— Presentation transcript:

1 Figure 2 Greatly reduced TDP2 protein and activity in 850-BR patient fibroblasts
Greatly reduced TDP2 protein and activity in 850-BR patient fibroblasts (A) TDP2 and actin protein levels in WCE (20 μg total protein) from 1-BR normal and 850-BR patient primary fibroblasts, as measured by immunoblotting. (B) TDP1 and TDP2 biochemical assay. Hydrolysis of a 3′-phosphotyrosyl (TDP1 assay) or 5′-phosphotyrosyl (TDP2 assay) bond releases the associated Cy5 fluorophore and alleviates quenching by the BHQ located on the opposite oligonucleotide terminus, resulting in elevated fluorescence at 640 nm. (C) Real-time (0–30 minutes) measurements of 5′-tyrosyl DNA phosphodiesterase activity (TDP2 assay) in reaction buffer (as a negative control) or WCE (15 μg protein) from 1-BR normal or 850-BR patient primary fibroblasts, using the single-stranded oligonucleotide (40 nM) with a 5′-phosphotyrosyl-linked Cy5 fluorophore and 3′-BHQ. (D) Real-time measurements (0–30 minutes) of 3′-tyrosyl DNA phosphodiesterase (TDP1 assay) conducted as above but using a 3′-phosphotyrosyl-linked Cy5 fluorophore and 5′-BHQ. Data are mean of 3 independent experiments ±SEM. Statistical significance was determined by two way analysis of variance (ANOVA). ***p < BHQ = black hole quencher; TDP2 = tyrosyl DNA phosphodiesterase 2; WCE = whole-cell extract. Guido Zagnoli-Vieira et al. Neurol Genet 2018;4:e262 Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.

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