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. 2010 Jul 21;285(40):30959–30970. doi: 10.1074/jbc.M110.138370

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 7. — Modulation of the association between activated type I TGF-β receptor and Smad proteins by MPK38. A and B, HEK293 cells were transfected with the indicated combinations of plasmid vectors expressing TβR1(TD), an activated type I TGF-β receptor, FLAG-Smad3 or -7, and GST-tagged wild-type (WT) or kinase-dead (K40R) MPK38, and the cell lysates were subjected to immunoprecipitation with an anti-HA antibody (α-HA). Complex formation between TβR1(TD) and Smad3 (A, left, top panel) or Smad7 (B, left, top panel) was determined by anti-FLAG antibody immunoblot. HEK293 cells were transfected with the indicated siRNA duplexes (MPK38-specific siRNA or control scrambled siRNA) together with plasmid vectors expressing TβR1(TD) and FLAG-Smad3 or -7. Complex formation between TβR1(TD) and Smad3 (A, right, top panel) or Smad7 (B, right, top panel) was determined by anti-FLAG antibody immunoblot. The expression level of endogenous MPK38 was determined by anti-MPK38 immunoblotting (A and B, right, bottom panels). C, effect of MPK38-mediated phosphorylation of Smad proteins on TβR1(TD)-Smads association. FLAG-tagged wild-type Smad proteins (Smad3 and -7) or their substitution mutants (S204A of Smad3 and T96A of Smad7) were cotransfected with HA-TβR1(TD) into HEK293 cells. The cell lysates were subjected to immunoprecipitation with an anti-HA antibody (α-HA), and complex formation between TβR1(TD) and Smad proteins was determined by an anti-FLAG antibody immunoblot (top panel). The relative level of complex formation between TβR1(TD) and Smad3 (A and C) or Smad7 (B and C) was quantitated by densitometric analysis, and -fold increase relative to control samples expressing TβR1(TD) and Smad3 or -7 alone was calculated. Sc, scrambled.