Fig. 5.

(A) PIASy does not inhibit Smad DNA-binding activity. Cell extracts from TGF-β-treated or untreated HaCaT cells were incubated with 2× SBE DNA probe in the absence or presence of increasing amount of purified Flag-PIASy protein (50, 100, 200, and 400 ng) and were then subjected to electrophoresis. (B) PIASy can be recruited to the SBE through interaction with Smads in DNA-affinity precipitation assay. (C) PIASy inhibits GAL4-Smad3 transcriptional activity. Mv1Lu/L17 cells were cotransfected with GAL4-Luc reporter gene, GAL4 DNA-binding domain GAL4 (DBD), or GAL4-Smad3, and increasing amount of T7-PIASy plasmid, treated with or without TGF-β, and analyzed for luciferase activity.