Abstract
CFTR functions as a chloride channel at the apical membrane of airway, gastrointestinal, and other epithelial cells. Immunofluorescence microscopy is commonly used to assess the subcellular localization and relative abundance of CFTR. Visualization of heterologously overexpressed CFTR is typically unproblematic and straightforward, whereas detection of small quantities of endogenous CFTR in tissues can be challenging and requires highly specific antibodies and optimized staining protocols. CFTR tagged by green fluorescent protein can be employed to study trafficking in live cells. Tagging of CFTR with an extracellular epitope permits detection exclusively at the cell surface and subsequent chasing allows visualization of endocytic trafficking.
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Acknowledgments
The authors gratefully thank the CF and non-CF volunteers for their tissue specimen donation; Richard Boucher for his comments, John R. Riordan and John Sheehan for their generous gifts of CFTR antibodies and MUC5AC polyclonal antibody, respectively; James Yankaskas and Marcus Mall for human tissue specimen collection; Scott Randell and Leslie Fulcher for providing the primary airway epithelial cells, Kim Burns, Tracy Barlotta, and Donald Joyner for their expert technical assistance; Lisa Brown for editing this manuscript; Michael Chua and Neal Kramarcy for microscopy assistance; and the UNC M. Hooker Microscopy Facility for making accessible their microscopes. This work is supported by CFF grants GENTZS04G0 and GENTZS07G0 (MG) and KREDA01I0 (SMK), the Mary Lynn Richardson Fund (SMK), and NIH Grants HL34322 and HL 51818-06A1 (SMK).
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Kreda, S.M., Gentzsch, M. (2011). Imaging CFTR Protein Localization in Cultured Cells and Tissues. In: Amaral, M., Kunzelmann, K. (eds) Cystic Fibrosis. Methods in Molecular Biology, vol 742. Humana Press. https://doi.org/10.1007/978-1-61779-120-8_2
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DOI: https://doi.org/10.1007/978-1-61779-120-8_2
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