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. Author manuscript; available in PMC: 2008 Mar 1.
Published in final edited form as: Biochim Biophys Acta. 2006 Dec 6;1773(3):427–439. doi: 10.1016/j.bbamcr.2006.11.017

Fig. 3. TGFβ1 and Smad3 promote vimentin reporter gene activity via the tandem AP-1 elements in COS-1 cells.

Fig. 3

(A) Western analysis of WCEs (40 μg) isolated from COS-1 cells transfected with vector alone (lane 1) or vector containing Smad2, Smad3, or Smad4 cDNAs. Antibodies used for immunoblotting (IB) are indicated and actin is included as a loading control. (B) The -775CAT construct was transiently transfected into COS-1 cells with empty vector or vector expressing Smad2, Smad3 or Smad4 as indicated. Reporter gene activity was measured as above. (C) Constructs -775CAT, -775mAP-1CAT, -725CAT, -353CAT, -319CAT, or -262CAT were transiently co-transfected with either empty vector or the Smad3 expression plasmid into COS-1 cells. Cells were harvested 48 h after transfection. Reporter gene activity was measured as above. (D) The -775CAT construct was transiently co-transfected into COS-1 cells with empty vector, or vector expressing Smad3 or deletion mutants thereof (as described in Fig. 2E), and Smad3 plus a constitutively active (TβRI-T204D) or dominant negative (TβRI-L45) TGFβ1 receptor. Reporter gene activity was measured as above. (E) Co-immunoprecipitation of Smad3 and c-Jun from COS-1 WCEs as described in Materials and Methods. COS-1 cells were transiently transfected with empty vector (lane 1) or Flag-Smad3 and c-Jun expression plasmids. Cells were harvested 48 h after transfection and protein expression verified in WCEs (40 μg) in lane 2. WCEs (1 mg) were immunoprecipitated with IgG or Flag-antibodies and immunoblotted (IB) with either c-Jun or Flag-Smad3 antibodies as indicated.