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The Impact of Different Normalization Methods on Western Blot Results

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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

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.

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  • 2021-05

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Impact of In-Utero Dexamethasone on Autonomic Regulation in Adulthood

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Premature babies are at risk of death from immature lung development. For this reason, pregnant mothers at risk for preterm delivery are administered dexamethasone (DEX), a synthetic glucocorticoid that promotes

Premature babies are at risk of death from immature lung development. For this reason, pregnant mothers at risk for preterm delivery are administered dexamethasone (DEX), a synthetic glucocorticoid that promotes fetal lung development. However, exposure to DEX in utero is associated with low birth weight and cardiovascular development pathologies. Moreover, our lab found that DEX administration in-utero leads to a sex-specific increase in stress-induced tachycardia in female, but not male offspring. This project seeks to expand on this preliminary finding of the heart by examining local effectors of activity from the sympathetic system (tyrosine hydroxylase and catechol-o-methyltransferase). Tyrosine hydroxylase was measured as it catalyzes the rate limiting step of norepinephrine synthesis while catechol-O- methyltransferase was studied as it catalyzes the degradation of norepinephrine. Acetylcholinesterase was used to measure parasympathetic activity as it catalyzes the degradation of the primary neurotransmitter of the parasympathetic nervous system, acetylcholine. Analyses of sympathetic as well as parasympathetic activity were done to determine influences of in-utero DEX exposure on autonomic regulation in adulthood. Pregnant rats were administered DEX (0.4 mg/kg, i.p.) or vehicle (20% w/v 2-hydroxypropyl ß- cyclodextran) at gestation days 18-21, with euthanasia of offspring occurring at around the time the offspring reached 13-15 weeks of age. Left ventricles and right atria were pulverized, processed and subjected to western blot analysis to determine expression of proteins of interest. Males exposed to DEX in-utero saw a decrease in tyrosine hydroxylase expression in left ventricle and right atrium when compared to vehicle control, a difference not seen with females. In addition, catechol-o-methyltransferase expression was increased in right atria from male, but not female rats. Acetylcholinesterase expression was reduced in the right atria of female, but not male rats. The present findings suggest reduced norepinephrine signaling in the heart of male, but not female DEX-exposed offspring. Given that we have previously found that female, but not male rats exhibit exaggerated stress-induced tachycardia, our current findings suggest that males possess a sex-specific compensatory mechanism allowing the heart to resist increased sympathetic signaling from the brain, one that females do not possess. The underlying mechanics of this proposed mechanism are unclear, and further investigation is needed in this subject to determine the significance of the findings from our study.

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  • 2021-05