The growth inhibition of dividing cells and most of the transcriptional responses upon TGF-β treatment depend on the Smad2, Smad3, and Smad4 transcription factors. These proteins shuttle continuously between the cytoplasm and the nucleus, transmitting the ligand status of the TGF-β receptors to the nuclear transcription machinery. In the absence of TGF-β ligand, Smads 2/3/4 reside predominantly in the cytoplasm. Following ligand binding to the TGF-β receptors, the dynamic equilibrium of shuttling Smads 2/3/4 shifts toward a predominantly nuclear state, where a high concentration of these transcription factors drives transcriptional activation and repression of genes required for proper cellular response. Here, we describe live cell imaging and immunofluorescence microscopy methods for tracking Smads subcellular localization in response to TGF-β and leptomycin B treatment. In addition, a method of fractionating nuclear and cytoplasmic proteins used to confirm the imaging results was presented. Our results support the notion that the R-Smad shuttling mechanism is distinct from Co-Smad.
