Reducing the amount of error and introduced data variability increases the accuracy of Western blot results. In this study, different methods of normalization for loading differences and data alignment were explored with respect to their impact on Western blot results. GAPDH was compared to the LI-COR Revert total protein stain as a loading control. The impact of normalizing data to a control condition, which is commonly done to align Western blot data distributed over several immunoblots, was also investigated. Specifically, this study addressed whether normalization to a small subset of distinct controls on each immunoblot increases pooled data variability compared to a larger set of controls. Protein expression data for NOX-2 and SOD-2 from a study investigating the protective role of the bradykinin type 1 receptor in angiotensin-II induced left ventricle remodeling were used to address these questions but are also discussed in the context of the original study. The comparison of GAPDH and Revert total protein stain as a loading control was done by assessing their correlation and comparing how they affected protein expression results. Additionally, the impact of treatment on GAPDH was investigated. To assess how normalization to different combinations of controls influences data variability, protein data were normalized to the average of 5 controls, the average of 2 controls, or an average vehicle and the results by treatment were compared. The results of this study demonstrated that GAPDH expression is not affected by angiotensin-II or bradykinin type 1 receptor antagonist R-954 and is a less sensitive loading control compared to Revert total protein stain. Normalization to the average of 5 controls tended to reduce pooled data variability compared to 2 controls. Lastly, the results of this study provided preliminary evidence that R-954 does not alter the expression of NOX-2 or SOD-2 to an expression profile that would be expected to explain the protection it confers against Ang-II induced left ventricle remodeling.

Damage to the Central Nervous System (CNS), such as traumatic brain injury (TBI) can often lead to a systemic inflammatory response since inflammatory mediators can be carried through the cardiovascular system. Past studies indicate that this inflammatory response that started at the CNS can increase the risk of heart disease. This growing interest in the heart-brain axis led our lab to explore if there is any impact of TBI on cardiac function and remodeling. TBI has been shown to have short-term effects on the heart, but few studies evaluate the long-term impact of TBI on the heart. To analyze any long-term impacts, we extracted hearts from rats 6 months post TBI, or sham that had been treated with vehicle or lipopolysaccharide (LPS) injections. LPS was administered to assess how inflammation could impact protein expression in the heart. Reactive oxygen species (ROS) targets such as NOX2, NOX4, SOD1, SOD2, catalase, and osteopontin were measured as potential indicators of cardiac remodeling. Rats that received vehicle TBI and LPS TBI resulted in no statistically significant differences (p>0.05) when evaluated as fold-change over the vehicle. This trend was consistent when normalizing to LPS sham. Since there were no changes in ROS targets, the hypothesis that there is long-term cardiac remodeling in the heart post-TBI was rejected. Further investigation is warranted since the present design of this study may not be ideal for evaluating long-term impact as histology samples were not obtained nor cardiac function assessments.