Abstract
Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays a central role in the response and adaptation to environmental and nutritional stimuli by initiating tissue-specific transcriptional reprogramming. Since its discovery in 1998, the field of PGC-1α biology has grown exponentially and a large body of research has elucidated the diverse roles of PGC-1α in brown adipose tissue thermogenesis, fatty acid oxidation, muscle fiber type switching, hepatic gluconeogenesis, and circadian clock regulation, etc. In addition, recent research has identified a splice variant(s) of PGC-1α in humans and rodents. The common misconception relating to PGC-1α is that it migrates at a predicted molecular weight of ~90 kDa by SDS-PAGE gel electrophoresis. However, several recent studies have provided solid evidence that the biologically relevant molecular weight of PGC-1α is ~110 kDa. In this chapter, we describe an optimized immunoblotting protocol that is developed to detect the low abundance protein PGC-1α and its alternatively spliced isoform named NT-PGC-1α in various rodent tissues. We also describe an optimized immunoprecipitation protocol that can isolate and concentrate endogenous PGC-1α and NT-PGC-1α. The protocols presented here will hopefully allow investigators to report accurate and reliable data regarding PGC-1α isoforms.
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Acknowledgments
The authors thank Jeho Shin for technical assistance and Ms. Cindi Tramonte for administrative support. This work was supported by the National Institutes of Health grants R01DK104748 (J.S.C.) and R01DK096311 (T.W.G.). The work used Cell Biology & Bioimaging and Genomics Core facilities that are supported in part by COBRE (NIH8 1P30GM118430-01) and NORC (NIH P30-DK072476) center grants from the National Institutes of Health.
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Gettys, T.W., Chang, J.S. (2019). An Optimized Immunoblotting Protocol for Accurate Detection of Endogenous PGC-1α Isoforms in Various Rodent Tissues. In: Badr, M. (eds) Nuclear Receptors. Methods in Molecular Biology, vol 1966. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9195-2_2
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DOI: https://doi.org/10.1007/978-1-4939-9195-2_2
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