Tax impairs TGF-β1–stimulated Smad3 DNA binding activity.
(A) An EMSA was performed by using a 32P-labeled probe derived from the PAI-1 promoter containing 3 CAGAC sequences and 10 μg of nuclear extracts from HepG2 cells transfected, with the Tax (Tax) or an empty expression vector (control), and induced (+) or not (−) for 30 minutes by TGF-β1. TGF-β1–induced complexes are indicated by arrows. Fifty molar excess of non–radio-labeled CAGAC sequence was added as competitor in 50 × molar excess (comp). Specific anti-Smad3 antibody (α-Smad3) was incubated before mixing with the CAGA probe. * indicates Smad3/4 complex. (B) HepG2 nuclear extracts used in (A) were mixed with a synthetic and palindromic CAGATCTG sequence. * indicates Smad3/4 complex. (C) A specific NF-κB probe derived from the IL-8 promoter was used with nuclear extract from MT2 cell line (MT2) or HepG2 cells transfected with a Tax expression vector (Tax) or an empty vector (control). ★ indicates NF-κB.
