Analysis of Caveolin in Primary Cilia
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Recent evidence has indicated that caveolins are localized at the base of primary cilia, which are microtubule-based sensory organelles present on the cell surface, and that Caveolin-1 (CAV1) plays important roles in regulating ciliary membrane composition and function. Here we describe methods to analyze the localization and function of CAV1 in primary cilia of cultured mammalian cells. These include methods for culturing and transfecting mammalian cells with a CAV1-encoding plasmid or small interfering RNA (siRNA), analysis of mammalian cells by immunofluorescence microscopy (IFM) with antibodies against ciliary markers and CAV1, as well as methods for analyzing ciliary CAV1 function in siRNA-treated cells by IFM and cell-based signaling assays.
Key wordsMammalian cell culture Primary cilia Caveolin Immunofluorescence microscopy siRNA-mediated knockdown qPCR
The authors acknowledge funding from the Independent Research Fund Denmark (6108-00457B and 8020-00162B), the Novo Nordisk Foundation (NNF14OC0011535, NNF15OC0016886 and NNF18OC0053024), Brødrene Hartmanns Fond (A31662), Kræftens Bekæmpelse (R146-A9590-16-S2), Carlsberg Foundation (CF18-0294), and the University of Copenhagen Excellence Programme for Interdisciplinary Research (2016 Funds). JBM and SKM were partially supported by PhD fellowships from the Department of Biology, University of Copenhagen.
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