Supplemental Figures

Three supplemental figures in JPG format.

Files in this Data Supplement:

• Appendix Figure 1 - Relationship between the amounts of protein loaded per lane and the net intensity for the phospholamban (PLB) immunoreactive bands. PLB immunoreactivity increased as the amount of protein loaded per lane increased (Panel A). The net intensity of these bands were analyzed and plotted on a scatterplot relative to the amount of protein loaded (Panel B). There was a significant linear relationship between the amount of protein loaded in a lane and the net intensity of the immunoreactive band. This procedure was repeated for all proteins of interest. Based upon these results, it was determined that loading 10 μg of protein per lane was necessary to accurately measure physiologic changes in protein abundance for all proteins of interest.
• Appendix Figure 2 - Relationship between the concentration of mRNA and cycle threshold (C_t) data measured by Real-Time PCR. The Real-Time PCR results using sarco(endo)plasmic reticulum calcium ATPase (SERCA) primers (Panel A) shows increases in C_t values with each 10-fold decrease in cDNA concentration. The measured C_t values were plotted on a scatterplot relative to the log of cDNA dilution factor (Panel B). There was a significant linear relationship between the amount of cDNA used and the C_t value. Based upon the slope of the relationship, the efficiency of the PCR reaction was determined. In this example, the PCR efficiency for the SERCA primers was 97.6 %. The average Real-Time PCR reaction efficiency for all primers sets used was 95 ± 2%.
• Appendix Figure 3 - Real-Time PCR melt curve data (Panel A) and 3% agarose gel image (Panel B) for determining primer specificity and product size. The Real-Time PCR melt curve data identifies one peak with a melting temperature of 87°C for all reaction wells. This indicates the presence of only one PCR product using sarco(endo)plasmic reticulum calcium ATPase (SERCA) primers. The ethidium bromide imaged agarose gel of Real-Time PCR products for each gene of interest identify only one PCR product for α-actin (actin), SERCA, phospholamban (PLB), and sodium calcium exchanger (NCX). In addition, no bands were visualized in the blank indicating that primer-dimers were not being produced. Lastly, size determination for each product based upon the molecular rulers (2000-50 bp) corresponds to the predicted product size for each of the designed pairs of PCR primers.