TWSG1 inhibits BMP-mediated Smad phosphorylation. HuH-7 cells were cultured in the presence or absence of 1000 ng/mL TWSG1, 10 ng/mL BMP2, or both for 1 hour. Total cell lysates (20 μg/lane) were immunoblotted using a rabbit antiserum specific to human phosphorylated-Smad1/5/8 (p-Smad1/5/8). Anti–total Smad1 antibody and anti–β-actin antibody were used as internal controls.