Luciferase and β-Galactosidase Assay for Activin-βA and AB2 Chimeras

HEK293T cells [53] (link) were seeded into 24-well plates coated with polylysine at a density of 150,000 cells/well. After 24 h cells were transfected overnight with a mixture of A3 Lux (25 ng) and β-galactosidase (25 ng) reporter plasmids, the transcription factor FAST2 (50 ng), and empty pCDNA3 vector (400 ng) using Perfectin® transfection reagent (GenLantis) according to the manufacturer's recommendations. Then the cells were treated with increasing doses of activin- βA or AB2 chimeras for 16–24 h. The cells were harvested in ice-cold lysis buffer (1% Triton X-100 in 25 mM glycylglycine, 4 nM EGTA, 15 mM MgSO₄ containing 1 mM dithiothreitol) and assayed for luciferase and β-galactosidase activities using standard methods. To assess the ability of the AB2 chimeras to bind known TGF-β co-receptors, the HEK293T cells were treated with increasing doses of Activin-βA or AB2 chimeras for 16–24 h in the presence or absence of transfected Cripto (mouse Cripto construct was a generous gift from Malcolm Whitman (Department of Cell Biology, Harvard Medical School, Boston, MA). Activity was then measured as previously described [23] .