5 March 2015 Development of confocal immunofluorescence FRET microscopy to Investigate eNOS and GSNOR localization and interaction in pulmonary endothelial cells
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Abstract
Confocal FRET microscopy is a widely used technique for studying protein-protein interactions in live or fixed cells. Endothelial nitric oxide synthase (eNOS) and S-nitrosoglutathione reductase (GSNOR) are enzymes involved in regulating the bioavailability of S-nitrosothiols (SNOs) in the pulmonary endothelium and have roles in the development of pulmonary arterial hypertension. Labeling of endogenous proteins to better understand a disease process can be challenging. We have used immunofluorescence to detect endogenous eNOS and GSNOR in primary pulmonary endothelial cells to co-localize these proteins as well as to study their interaction by FRET. The challenge has been in selecting the right immunofluorescence labeling condition, right antibody, the right blocking reagent, the right FRET pair and eliminating cross-reactivity of secondary antibodies. We have used Alexa488 and Alexa568 as a FRET pair. After a series of optimizations, the data from Confocal Laser Scanning Microscopy (CLSM) demonstrate co-localization of eNOS and GSNOR in the perinuclear region of the pulmonary endothelial cell primarily within the cis-Golgi with lower levels of co-localization seen within the trans-Golgi. FRET studies demonstrate, for the first time, interaction between eNOS and GSNOR in both murine and bovine pulmonary endothelial cells. Further characterization of eNOSGSNOR interaction and the subcellular location of this interaction will provide mechanistic insight into the importance of S-nitrosothiol signaling in pulmonary biology, physiology and pathology.
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Shagufta Rehman, Shagufta Rehman, Kathleen Brown-Steinke, Kathleen Brown-Steinke, Lisa Palmer, Lisa Palmer, Ammasi Periasamy, Ammasi Periasamy, } "Development of confocal immunofluorescence FRET microscopy to Investigate eNOS and GSNOR localization and interaction in pulmonary endothelial cells", Proc. SPIE 9329, Multiphoton Microscopy in the Biomedical Sciences XV, 93290G (5 March 2015); doi: 10.1117/12.2180163; https://doi.org/10.1117/12.2180163
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