Effect of HNF-1β ectopic expression on TGF-β1-induced phosphorylation of Smad3 in hRPTECs.

Human RPTECs were stimulated with 3 ng/ml TGF-β1 for 48 h followed by re-stimulation with fresh TGF-β1 for 12 h or 24 h. After replacement with fresh TGF-β1, the hRPTECs were infected with 1.0 MOI and 2.0 MOI Ad-HNF1B or Ad-LacZ. Representative western blotting shows the expression of phosphorylated Smad3 and total Smad3 at 48 kD in Ad-HNF1B-transfected hRPTECs 12 (A) and 24 h (B) after TGF-β1 re-stimulation. Densitometric quantification of the corresponding bands was performed using an image analyzer. The data are presented after normalization to total Smad3 expression. Each column presents the means of twice independent experiments from non-stimulation with Ad-LacZ (white), TGF-β1 stimulation for 48 h followed by incubation with TGF-β1 and Ad-LacZ (hatched line), and TGF-β1 and Ad-HNF1B (dot) for 12 (C) and 24 h (D). Each dot symbol shows an individual value.