Figure 7.

Smad3 and Smad4 together bind to the PE2.1 element of the PAI-1 promoter. (A) Bosc23 cells were transfected as described in Materials and Methods; cells in each dish were transfected with 2 μg of plasmid encoding Flag-N-Smad3 or Smad4 (pEXL–Smad4), and 1 μg of plasmid encoding TβRI-KR (pCMV5–TβRI–KR) or TβRI-T204D (pCMV5-TβRI-T204D) as indicated. The total amount of DNA for each dish was adjusted to 5 μg with pEXL–GFP. The gel-shift assay at top was carried out with the ³²P-labeled probe and 1 μl of cell lysate as described in Materials and Methods, and exposed to a Fuji PhosphorImager plate. The minor lower band in lane 12 at top probably represents Smad3 and Smad4 protein binding to only one of the two tandem repeats of the PE2.1 element. (Middle, bottom) Immunoblots with 5 μl (150 μg of proteins) of cell lysates in each lane that were blotted with an anti-Flag M2 antibody, recognizing the Flag epitope-tagged Smad3, and with an anti-Smad4 antibody, respectively, as described in Materials and Methods. As indicated, the levels of expression of Smad4 were the same in all cases (lanes 3,4,7,8,11,12) as were those of the Flag-tagged Smad3 (lanes 2,4,6,8,10,12). (B) Cell lysates containing both Smad4 and the Flag-tagged Smad3 were incubated with ³²P-labeled PE2.1 DNA, followed by addition of 1 μl of an irrelevant control antibody or preimmune serum (lanes 2,4) or the anti-Flag antibody (anti-Smad3) (lane 3) and the anti-Smad4 antibody (lane 5), followed by gel electrophoresis as described in Materials and Methods.