TGF-β represses AFP mRNA. (a) AFP mRNA expression was inhibited by ALK5ca in HuH-7 cells. The cells were infected with LacZ (left lane) or ALK5ca-expressing adenoviruses (right lane) at a dose of 250 multiplicity of infection. After 42 hours of infection, total RNA and cell lysates were prepared for RT-PCR analysis (a) and Western blot analysis (IB) (b), respectively. (a) Expression of AFP, TMEPAI, and β-actin mRNA was detected by RT-PCR. TMEPAI which is a direct target gene of TGF-β signal [29–31] was used as a positive control. (b) For IB, antiphosphorylated Smad2 (PS2), anti-ALK5, and anti-β-actin antibodies were used. (c) Expression of AFP mRNA upon TGF-β stimulation. HuH-7 cells were treated with 5 ng/mL TGF-β for the indicated times. Then, RT-PCR was performed as described in (a). Each PCR condition is described in Table 1.