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. Author manuscript; available in PMC: 2015 Oct 1.
Published in final edited form as: Cell Signal. 2014 Jun 3;26(10):2276–2283. doi: 10.1016/j.cellsig.2014.05.018

Figure 7.

Figure 7

EGF induces the phosphorylation and expression of TGIF. (A) HKCs were treated with EGF (25 ng/ml) for indicated periods of time. Whole cell lysates were examined for TGIF using an antibody that recognizes total TGIF. β-actin was used as a loading control. (B) Densitometric analysis of pTGIF was normalized to β-actin and expressed as fold change over control. *p<0.05 compared with control (n=3). (C) Cells were pretreated with the inhibitors, wortmannin (W, 50 nM), AG1478 (AG, 5 μM), SP600125 (SP, 20 μM) and PD0325901 (PD, 100 nM) for 1 hour before 30 min treatment with EGF (25 ng/ml). Whole cell lysates were examined for TGIF by western blot. β-actin was used as a loading control. (D) Densitometric analysis of pTGIF was normalized to β-actin and expressed as fold change over DMSO control. *p<0.05 compared with DMSO control (n=3). (E) Cells were treated with EGF (25 ng/ml) for 3 days. Whole cell lysates were examined for total TGIF. Tubulin was used as a loading control. The vertical line in the middle of the blot indicates the removal of irrelevant lanes within the same blot. (F) Densitometric analysis of total TGIF was normalized to tubulin and expressed as fold change over control. *p<0.05 compared with control (n=3). (G) Cells were treated with TGF-β (1 ng/ml) and/or EGF (25 ng/ml) for 24 hours. Quantitative PCR was performed to assess the mRNA level of TGIF. (H) A wild type TGIF construct (wt TGIF) was overexpressed in HKCs along with αSMA promoter reporter construct (SMALuc). 3 hours after transfection, cells were exposed to 1 ng/ml TGF-β for another 24 hours. The luciferase activity assayed in triplicates was normalized to β-galactosidase (β-gal) to control transfection efficiency. * p<0.05 compared with vehicle control (n=3). Western blots confirmed the overexpression of TGIF. (I) HKCs were pretreated with DMSO or PD0325901 (100 nM) for 1 hour, then incubated with EGF (25 ng/ml) for 72 hours. TGIF protein expression was examined by western blot. β-actin was used as a loading control. (J) Densitometric analysis of total TGIF was normalized to β-actin and expressed as fold change over DMSO control. *p<0.05 compared with non-EGF-treated control (n=3).