Quantitative analysis of mitochondrial ETC enzymes, myoglobin (MB) and MTHFD2 in cardiac mitochondria of TazKD mice.

<p>(A) Quantification of Taz mRNA levels in hearts of 3 weeks old (P21) and 3 months old (3M) WT and TazKD mice. The expression levels were determined by quantitative RT-PCR and represented as relative normalized expression. (B) Representative Western blots of mitochondrial proteins from TazKD and WT hearts. Mitochondrial proteins (30 μg) were separated by SDS-electrophoresis and subjected to Western blot analysis using a cocktail of monoclonal antibodies specific to individual polypeptides of ETC complexes NDUFA9 (C-I), SDHB (C-II), UQCRC (C-III), COX4 (C-IV) and ATP5A (C-V). Additionally, blots were probed with antibodies specific to Rieske Fe-S cluster protein (UQCRFS1) and myoglobin (MB). Myoglobin content was analyzed in mitochondrial fraction (Mito) and the total cardiac tissue protein extract (T.E.) fractions. Antibodies specific to mitochondrial malate dehydrogenase (MDH) were used as a loading control. The entire (uncropped) images of Western blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128561#pone.0128561.s001" target="_blank">S1 Fig</a>. (C) Quantitative assessments of Western blot analyses. Bars represent fold-changes in band intensities in TazKD samples compared to WT controls. For <b>A</b> and <b>C:</b> Data shown are means ± standard error of mean (SEM). Number of experiments per group is denoted in the corresponding bar. Asterisks indicate significant differences of P≤0.05 (Student’s t-test) between WT and TazKD.