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. 2007 Sep 10;178(6):981–994. doi: 10.1083/jcb.200703106

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Copyright © 2007, The Rockefeller University Press

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Figure 7. — Interaction of Smads with Imp7 and Imp8, and the regulation by Ran-GTP. (A) Co-immunoprecipitation of Smad1 with Imp7 and Imp8. 293T cells were transfected with indicated expression plasmids and the whole-cell extract (WCE) was immunoprecipitated with anti-Flag antibody. Protein A/G bead was used as the control (c). The bound proteins and the input extract (WCE) were analyzed by immunoblotting as indicated. (B) Co-immunoprecipitation of Smad2 with Imp7 and Imp8. Same experimental design as in A, but with different expression plasmids transfected as indicated. (C) Mapping of Smad3 domains involved in interaction with Imp7/8. Recombinant GST fusions of indicated Smad3 or Smad2 fragments were used to pull down endogenous Imp7/8 in HeLa cells. The bound proteins were analyzed by an antibody that recognized both Imp7 and 8. Comparable amount of GST proteins was used in the pull down as judged by the Coomassie stain intensity. The arrowheads mark GST fusion proteins on the SDS-PAGE gel. S3MH1: aa 1–155; S2MH1: aa 1–185; S3MH2: aa 231–425; S3(L+MH2): aa 145–425; S3FL: full-length. Schematic drawing of Smad3 is also shown. (D) Purified GST-Imp8 on glutathione beads was used to pull down purified recombinant Smad1 and Smad3. The bound proteins were examined by immunoblotting using indicated antibodies. GST was used as the control. (E) Ran-GTP interrupts association between Smad3 and Imp8. GST-fusion of full-length Smad3 (GST-S3FL) was used in a pull-down experiment as in C. The bound proteins were further incubated with RanQ69L-GTP or BSA, and proteins released into the supernatant were collected and analyzed at indicated time points (15 min and 45 min elution). At the 45-min time point, the beads were washed again and proteins remaining bound to GST-S3FL (bound) were also examined by anti-HA immunoblotting.