The transforming growth factor beta (TGFβ) signaling pathway is important for normal cell homeostasis and has critical roles in apoptosis, cell-cycle arrest, and cellular differentiation (reviewed in Massague, 2008). In the classical TGFβ pathway, the endosomal trafficking of receptors has a direct outcome on signal transduction-receptors internalized via clathrin-mediated endocytosis enter the early endosome and propagate signaling, while those internalized via membrane rafts are targeted for degradation. Recently, there have been a number of articles that have identified TGFβ receptor-binding proteins that direct receptor endocytosis and/or intracellular trafficking and affect signal output (Atfi et al., 2007; Bauge, Girard, Leclercq, Galera, & Boumediene, 2012; Bizet et al., 2011, 2012; Chen et al., 2007; Gunaratne, Benchabane, & Di Guglielmo, 2012; Hao et al., 2011; McLean, Bhattacharya, & Di Guglielmo, 2013; Zhao et al., 2012). Given the importance of TGFβ receptor trafficking to signaling outcome, this chapter will focus on strategies to isolate membrane rafts and techniques to follow the trafficking of cell-surface TGFβ receptors and provide examples of functional readouts to assess TGFβ signal transduction.
Keywords: Immunofluorescence microscopy; Membrane rafts; Receptor internalization; Subcellular fractionation; Transforming growth factor beta.
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