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. Author manuscript; available in PMC: 2012 May 1.
Published in final edited form as: J Cell Physiol. 2011 May;226(5):1415–1424. doi: 10.1002/jcp.22477

Fig. 5. TGFβ treatment increase binding Smad4 protein with p14ink4b promoter.

Fig. 5

A. Chromatin was isolated from TGFβ1 (5ng/ml) or TGFβ3 (5ng/ml) treated/untreated MEE cells, immunoprecipitated with anti Smad4 antibody (overnight) and analyzed by quantitative real-time PCR with primers recognizing A, B and C Smad4 binding elements in p15ink4b promoter. Serial dilutions of pGL3-p15wild type plasmid were used as quantitative standards. Both TGFβ1 and TGFβ3 increase binding of Smad4 with SBE in the p15ink4b promoter. TGFβ1 induces binding of Smab4 with all three SBE in variable degree, whereas TGFβ3 increases only binding with SBE B.

B. Chromatin was isolated from TGFβ1 (5ng/ml) or TGFβ3 (5ng/ml) treated/untreated MEE cells, immunoprecipitated with anti Smad4 antibody (overnight) and analyzed by quantitative real-time PCR with primers recognizing A, B and C Smad4 binding elements in p15ink4b promoter. Serial dilutions of pGL3-p15wild type plasmid were used as quantitative standards. Both TGFβ1 and TGFβ3 increase binding of Smad4 with SBE in the p15ink4b promoter. TGFβ1 induces binding of Smab4 with all three SBE in variable degree, whereas TGFβ3 increases only binding with SBE B. The results are shown as a mean ± SD obtained from three independent chromatin preparations. Asterisk indicates p ≤ 0.05, double asterisk – p ≤ 0.005.

C. Input control for quantitative real-time PCR of immunoprecipitated chromatin. Chromatin was isolated as described in A and analyzed by quantitative PCR with primers recognizing A, B and C Smad4 binding elements in p15ink4b promoter with DNA standards described in A. Primers recognizing B and C Smad4 binding elements display higher efficiency than primers specific for SBE A, however statistically significant differences between TGFβ1-, TGFβ3-treated or untreated samples are not detected (p ≥ 0.05). Error bars show the standard deviation of three independent chromatin preparations.