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Expression and activity of superoxide dismutases in chagasic mice.

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posted on 2018-07-25, 17:28 authored by Jake J. Wen, Nisha Jain Garg

C57BL/6 (WT and MnSOD+/-) mice were infected with Trypanosoma cruzi (10,000 Tc/mouse), and sacrificed at 150 days’ post-infection (pi) corresponding to chronic disease phase. (A) Total DNA was used as template for genotyping the MnSOD+/- (vs. WT) mice. The amplification of a 229 bp band indicates the integration of xx within MnSOD gene (B-D) Reverse transcription—quantitative PCR (RT-qPCR) evaluation of myocardial levels of SOD1 (B), SOD2 (C) and SOD3 (D) mRNAs was performed in WT and MnSOD+/- mice before infection and at 150 days pi. The data were normalized to GAPDH mRNA (n = 6 mice per group, triplicate observations per mouse). (E&F) Representative Western blot images for myocardial levels of MnSOD and GAPDH (loading control) are shown (E, n = 3 mice per group). Densitometry analysis was performed for Western blot gels representing n = 6 mice per group (duplicate observations per mouse), and data were normalized to GAPDH levels (F). (G) Heart homogenates were subjected to differential centrifugation to isolate mitochondria. Mitochondrial MnSOD activity was determined by a spectrophotometric assay (n = 6 mice per group, duplicate observations per mouse). Data in bar graphs are plotted as mean value ± SEM. Statistical significance are marked as aMnSOD+/- vs. WT, bWT.Tc vs. WT, cMnSOD+/-.Tc vs. MnSOD+/-, dMnSOD+/-.Tc vs. WT.Tc, and eMnSOD+/-.Tc vs. WT (p value < 0.05).