Figure 1.

In vitro import assay for Smad3. (A) Profile of bacterially purified Smad3 (lane 1) and Smad3D proteins (lane 2) after SDS-PAGE and Coomassie Brilliant Blue staining. (B) Microinjection of bacterial Smad3 together with bacterial GSTNLSGFP protein in the cytoplasm of human colon carcinoma SW480.7 cells. The cells were previously transiently infected with adenoviruses that encode for LacZ (control) or caALK-5. The caALK-5–infected cells were also treated with TGF-β1 (+TGF-β) immediately after microinjection and for 1 h. (C) In vitro import of Smad3, Smad3D, and GSTNLSGFP in the absence (−) and presence (+) of cytosol in digitonin-permeabilized HeLa cells. In this assay, each protein was added at 0.5 μM. Smad proteins were detected by anti-Flag/Cy3 immunofluorescence and GSTNLSGFP by green autofluorescence. Nuclear morphology was assessed by phase contrast microscopy.