Fig. 4. Characterization of in vitro cell-based Dpp signaling assay. (A) Dose dependency of Dpp in the signaling assay was investigated. S2 cells expressing Flag-Mad proteins were incubated with the indicated amounts of Dpp protein for 3 hours at 25°C, and the cell extracts were blotted and analyzed by western blotting using anti-phosphoMad or anti-Flag antibody. Dpp activity was detected at as low as 10^-10 M Dpp, and at 10^-9 M Dpp, the signals were saturated. (B) Dpp signals through Tkv as a type I receptor and Punt as a type II receptor in S2 cells. S2 cells were transfected with Flag-Mad for control (C), or Flag-Mad and dsRNA of tkv, sax, or punt (R). Three days after transfection, cells were collected split into two fractions, one of which was used for a phosphoMad signaling assay and the other was used for RT-PCR.