Fig. 4. Physical association of Smad2, Smad3 and Smad4 with Sp1. (A) TGF-β-dependent association of Smads and Sp1 in vivo. HaCaT cells were transfected with HA-tagged Smads or CBP, with (+) or without (–) an expression plasmid for activated TβRI. Cell lysates were immunoprecipitated (IP) with an anti-HA antibody, followed by immunoblotting (IB) with an anti-Sp1 antibody to detect Smad-bound Sp1 (upper panel). Cell lysates were also directly immunoblotted with anti-Sp1 or anti-HA antibodies to demonstrate expression of endogenous Sp1 (middle panel) or transfected Smads (lower panel). (B) Smad3 co-immunoprecipitates with endogenous Sp1 in the presence of Smad2 or Smad4. HaCaT cells were transfected with expression plasmids for Flag-tagged Smad3 and HA-tagged Smad2 or 4. Immunoprecipitation with anti-Flag antibody was followed by anti-Sp1 immunoblotting to detect Smad3-bound Sp1. Expression levels of Flag-tagged Smads (middle panel) or HA-tagged Smads (lower panel) were shown by anti-Flag or anti-HA immunoblotting. (C) Direct interaction of GST–Sp1(1–621) with ³⁵S-labeled Smad2 and its segments. Smad2N, Smad2NL, Smad2LC and Smad2C cover the Smad2 regions of amino acids 2–183, 2–273, 181–467 and 270–467, respectively. (D) Direct interaction of GST–Sp1(1–621) with ³⁵S-labeled Smad4 and its segments. Smad4N, Smad4NL, Smad4LC and Smad4C cover the Smad4 regions of amino acids 2–154, 2–300, 141–552 and 294–552, respectively.