TGF-β1 phosphorylates SMAD5 in normal and malignant mature B cells. (A) Mature B cells were purified from the spleens of 3 WT C57BL/6 mice and exposed to TGF-β1 (5 ng/mL) or vehicle control for 4 hours. Western blot analysis demonstrates prompt induction of SMAD1/5 phosphorylation in these cells. (B) TGF-β1 (5 ng/mL, 1 hour) also induced marked activation of SMAD1/5 in DLBCL cell lines. In both instances, the western blotting was carried out with nuclear extracts. Equal loading is verified with immunoblotting to total SMAD5. The assays shown in panel A represent 3 biological replicates (B cells from 3 mice), and the assays shown in panel B were confirmed in multiple (more than 3) biological replicates.