Skip to main content
. 2014 Feb 26;35(6):1379–1388. doi: 10.1093/carcin/bgu050

Fig. 4.

Fig. 4.

Six1 overexpression in HKc/DR activates Smad-independent pathways. (A) Phospho-Smad2/3 was detected by western blot analysis in cell extracts prepared from HKc/DR-Ctrl (DR-Ctrl) and HKc/DR-Six1 (DR-Six1). β-Actin was used as loading control. (B) Upper panel: HKc/DR-Ctrl and HKc/DR-Six1 were cotransfected with a TGF-β-inducible luciferase reporter (p6SBE-luc), which contains six tandem copies of the SBE, and pRL-SV40 Renilla luciferase, as control for transfection efficiency. Luciferase activity was determined 48h after transfection. Firefly luciferase values are normalized to Renilla luciferase. Bars indicate standard deviation. Middle/lower panels: HKc/DR-Ctrl or HKc/DR-Six1 were transfected with either p6SBE-luc or p6SME-luc, which contains mutated SBEs (SME). Twenty-four hours after transfection, cells were treated with 40 pM TGF-β1 for 24h and then luciferase activity was determined. Values are normalized to Renilla luciferase. Bars indicate standard deviation. (C) Protein levels of p-ERK, ERK, p-P38, P38, p-JNK and JNK were determined by western blotting of cell lysates prepared from HKc/DR-Ctrl (DR-Ctrl) and HKc/DR-Six1 (DR-Six1). β-Actin was used as a loading control. (D) Western blot of p-AKT (Tyr326), p-AKT (Ser473) and p-PI3-kinase p85 (Tyr467/199) in HKc/DR-Ctrl (DR-Ctrl) and HKc/DR-Six1 (DR-Six1). β-Actin was used as a loading control.