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. 2001 Apr;12(4):1079–1091. doi: 10.1091/mbc.12.4.1079

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Copyright © 2001, The American Society for Cell Biology

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Smad3 interacts with importin-β1 in an activation/phosphorylation-dependent manner. (A-C) SW480.7 and CCL64 cells were used giving essentially identical results. Representative immunoblots are shown in this figure. (A) In vitro interaction assay between GST (lane 1), GST-importin-β1 (GST-β1, lane 2), GST-importin-β2 (GST-β2, lane 3) and Smad3. Detergent extracts from transiently coinfected cells with adenoviruses encoding LacZ and Smad3 or caALK-5 and Smad3 and treated (+TGF-β) or not (LacZ) with TGF-β1 were isolated. Extracts were incubated with the three GST protein affinity columns and the bound Smad3 was analyzed by immunoblotting (IB) with anti-Flag antibody. In lane 4 an aliquot of the total cell lysate was analyzed by immunoblotting directly. (B) In vitro interaction assay between GST (lane 1), GST-importin-α1 (GST-α1, lane 2), GST-importin-α2 (GST-α2, lane 3), GST-importin-α3 (GST-α3, lane 4) and GST-importin-β1 (GST-β1, lane 5) and Smad3. Cell extracts were prepared and analyzed as in A. The total lysate control is in lane 6. (C) Specificity of the Smad3–importin-β1 interaction. In vitro interaction assay between GST-importin-β1 (GST-β1, all lanes) and Smad3. Cell extracts treated as indicated on top of the panel were prepared and analyzed as in A by using anti-Flag (first panel) or anti-phospho-Smad (second panel) immunoblotting. Analysis of total lysate aliquots (Total) with the same two antibodies is shown in the third and fourth panels, respectively. Smad7 was also detected by anti-Flag immunoblotting (fifth panel). Asterisks on the right of the second and fourth panels indicate nonspecific bands resulting from the rabbit anti-phospho-Smad serum. (D) C-Terminal phosphorylation-dependent interaction between Smad3 and importin-β1. In vitro interaction assay between GST (lanes 1 and 2), GST-importin-β1 (GST-β1, lanes 3 and 4) and histidine-tagged baculoviral Smad3 (S3, lanes 1 and 3), or C terminally phosphorylated Smad3 (S3P, lanes 2 and 4). Bound Smad3 proteins were analyzed by immunoblotting by using anti-histidine (His) antibody. Aliquots of the baculoviral proteins were separately analyzed by sequential anti-histidine (lanes 5 and 6) and anti-phospho-serine (P-Ser, lanes 7 and 8) immunoblotting. (A–D) Position of the relevant protein bands is indicated on the sides of the panels.

Smad3 interacts with importin-β1 in an activation/phosphorylation-dependent manner. (A-C) SW480.7 and CCL64 cells were used giving essentially identical results. Representative immunoblots are shown in this figure. (A) In vitro interaction assay between GST (lane 1), GST-importin-β1 (GST-β1, lane 2), GST-importin-β2 (GST-β2, lane 3) and Smad3. Detergent extracts from transiently coinfected cells with adenoviruses encoding LacZ and Smad3 or caALK-5 and Smad3 and treated (+TGF-β) or not (LacZ) with TGF-β1 were isolated. Extracts were incubated with the three GST protein affinity columns and the bound Smad3 was analyzed by immunoblotting (IB) with anti-Flag antibody. In lane 4 an aliquot of the total cell lysate was analyzed by immunoblotting directly. (B) In vitro interaction assay between GST (lane 1), GST-importin-α1 (GST-α1, lane 2), GST-importin-α2 (GST-α2, lane 3), GST-importin-α3 (GST-α3, lane 4) and GST-importin-β1 (GST-β1, lane 5) and Smad3. Cell extracts were prepared and analyzed as in A. The total lysate control is in lane 6. (C) Specificity of the Smad3–importin-β1 interaction. In vitro interaction assay between GST-importin-β1 (GST-β1, all lanes) and Smad3. Cell extracts treated as indicated on top of the panel were prepared and analyzed as in A by using anti-Flag (first panel) or anti-phospho-Smad (second panel) immunoblotting. Analysis of total lysate aliquots (Total) with the same two antibodies is shown in the third and fourth panels, respectively. Smad7 was also detected by anti-Flag immunoblotting (fifth panel). Asterisks on the right of the second and fourth panels indicate nonspecific bands resulting from the rabbit anti-phospho-Smad serum. (D) C-Terminal phosphorylation-dependent interaction between Smad3 and importin-β1. In vitro interaction assay between GST (lanes 1 and 2), GST-importin-β1 (GST-β1, lanes 3 and 4) and histidine-tagged baculoviral Smad3 (S3, lanes 1 and 3), or C terminally phosphorylated Smad3 (S3P, lanes 2 and 4). Bound Smad3 proteins were analyzed by immunoblotting by using anti-histidine (His) antibody. Aliquots of the baculoviral proteins were separately analyzed by sequential anti-histidine (lanes 5 and 6) and anti-phospho-serine (P-Ser, lanes 7 and 8) immunoblotting. (A–D) Position of the relevant protein bands is indicated on the sides of the panels.