Figure 4

Validation of the most representatitve protein changes detected in ischemic myocardium after acute infarction. (a) Western blot analysis and corresponding quantitative densitometry of ATP5A, UQCRC2, SDHB, NDUFB8, UQCRFS1, COXIV and ATP5B confirming the decrease in mitochondrial protein content 7 days after reperfusion with respect to baseline. (b) Western blot analysis and quantitative densitometry of cardiac Myosin heavy chain, TNNT2 and TPM1 confirming the decrease in sarcomeric contractile proteins 7 days after reperfusion. Each lane of western blot corresponds to a protein extract obtained from one animal. Protein values are normalized relative to GAPDH. In the bar plots, results are shown as the mean ± s.e.m. of five determinations. *Indicates p value < 0.05; **indicates p value < 0.01 versus baseline by Mann-Whitney test. Full-length blots from panels a and b are presented in Supplementary Figure S8 as well as their raw integrated densities before and after normalization can be consulted in Supplementary Figures S9 and S10. iTRAQ quantifications of the proteins from panels a and b are shown in the Supplementary Figure S11a for comparison. (c) Label free individual protein quantifications of ATP5A, ATP5B, UQCRFS1, SDHB, MYH7, TNNT2 and TPM1 confirming the decrease in mitochondrial and contractile protein content. Protein quantifications are represented as log2(fold-change) of iBAQ intensities of individual animals from R-Day 7 compared with pooled average of iBAQ intensities from all Baseline group animals. (d) Label free quantitative proteomics confirming the decrease in mitochondrial and contractile proteins and the increase in acute-phase related proteins, collagens, extracellular matrix proteins (ECM), proteolysis related proteins and gene expression related protein contents, 7 days after reperfusion with respect to the baseline. Protein abundances were expressed as the iBAQ values. Results are shown as the mean ± s.e.m of five determinations performed in individual animals, *indicates FDR < 0.05; **indicates FDR < 0.01, ***indicates FDR < 0.001 versus baseline by Mann-Whitney test after Benjamini–Hochberg correction. Label free individual protein quantifications included in the mitochondrial, contractile, acute-phase, collagens, ECM, proteolysis and gene expression protein groups are shown in Supplementary Figure S13.