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. 2015 Aug 18;17(7):525–537. doi: 10.1016/j.neo.2015.06.004

Figure 4.

Figure 4

Kaempferol does not inhibit Smad3 C-terminal phosphorylation and its complex formation with Smad4 and translocation into the nucleus upon TGF-β1 stimulation. (A) A549 cells were pretreated with DMSO or 25 μM of KF for 30 minutes before treatment with 5 ng/ml of TGF-β1 for the indicated periods, after which the extent of Smad3 phosphorylation at Sre423/425 was determined by Western blot analysis. (B) A549 cells were pretreated with 25 μM of KF at the indicated concentrations for 30 minutes and then stimulated with 5 ng/ml of TGF-β1 for 1 hour. Western blot analysis was performed as in A. β-Actin levels were monitored as a loading control for whole extracts. (C) A549 cells were pretreated with DMSO or 25 μM of KF for 30 minutes before treatment with 5 ng/ml of TGF-β1 for 30 minutes, and cell lysates were immunoprecipitated with anti-Smad3 antibody and immunoblotted as indicated. β-Actin levels were monitored as a loading control for whole extracts. (D) A549 cells were treated as in A except for the TGF-β1 treatment for 10 and 90 minutes. (E) A549 cells were treated as in B. All cells were then subjected to immunofluorescence staining for Smad3 (green). DAPI (blue) was used to stain nuclei.