Activin-A and TGF-β receptor expression and activin-A–induced Smad signaling in NK cells. NK cells were purified from buffy coats of healthy donors and assessed for expression of activin type I (ALK4) and type II (ActRIIA and ActRIIB) or TGF-β type I (RI) and type II (RII) receptors mRNA by qRT-PCR (A-D). In brief, NK cells were lysed immediately after purification or after 16 hours of culture in media supplemented with IL-2 (20 U/mL) + IL-12 (10 ng/mL) with or without the addition of recombinant activin-A or TGF-β. RNA was then extracted and qRT-PCR performed. (E) Western blotting for detection of Smad 2/3 or β-actin expression. In brief, purified NK cells (5 × 10⁵) were cultured in 24-well plates for 4 hours with either IL-2 alone or with IL-2 and recombinant human activin-A (50 ng/mL). NK cells were then lysed and protein extracted. Data are shown as either one representative experiment from 3 separate donors (E) or the mean plus or minus 1 SD of 3 separate donors (A-D). *P < 0.01 and **P < .02 versus ex vivo levels.