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. Author manuscript; available in PMC: 2010 Jun 8.
Published in final edited form as: Biochem J. 2009 Apr 15;419(2):485–495. doi: 10.1042/BJ20081434

Figure 4. Interactions of Sp1, KLF6 and Smad3 based on co-immunoprecipitation.

Figure 4

(A) Cos-7 cells were transfected with expression vectors for Smad3 and Smad4 in the presence or absence of constitutively active TβRI (TβRI C. A.) (Cos cells have very low amounts of this receptor and of Smad proteins). GST–KLF6 fusion pull-down was generated from the protein extracts of transfected cells. As a control for specificity of the method, when Smad3 was not transfected (lane 3), only Sp1 was recovered by GST–KLF6 pull-down. (B) Co-immunoprecipitation (IP) experiments in HUVECs, either untreated (C) or after treatment with TGF-β (10ng/ml) and recovery from WH for 24 h (T). IgG is a control lane following immunoprecipitation using human IgG. (C) Co-immunoprecipitation experiments were carried out in Schneider cells, which do not express Sp1, or KLF6. Schneider cells were co-transfected with pPACSp1 and Flag Smad3 expression vectors on the left side of (B), or with pPACKLF6 and Flag Smad3 on the right side. When the immunoprecipitation was made using the Flag tag to recover Smad3, Sp1 could be revealed by Western Blot (WB) in the immunoprecipitate (lane 1, left side). However, when transfecting with pPACKLF6 and Smad3, in the Flag Smad3 immunoprecipitate we could not detect KLF6. In the upper part, Western blots (WBs) show the expression of the transfected proteins in all the cases of transfected proteins. Lane 3C (293T) is a control of the migration for KLF6. The co-immunoprecipitation experiments were repeated three times and the results are representative. (D) Sequential co-immunoprecipitation in HEK-293T cells. HEK-293T cells were doubly or triply transfected with KLF6 and Smad3, or KLF6, Smad 3 and Sp1 expression vectors respectively. Lane 1 shows the results obtained with double transfection of KLF6 and Smad3, whereas lane 2 displays the triple-transfection results. On the left-hand side, Western blots of total extracts are shown. On the right-hand side the results for the first immunoprecipitation using KLF6 antibody and the second immunoprecpipation with the anti-Flag tag are shown. There is a substantial enrichment of Smad3 in the ternary complex after the second immunoprecipitation, which is dependent on the transfection of Sp1.