Fig. 7. Tld reverses the Sog and Tsg synergistic inhibition of Dpp signaling in vitro. (A) The indicated combination of proteins, including 10^-9 M of Dpp, were pre-incubated for 6 hours and further incubated with S2 cells expressing Flag-Mad for 3 hours. The cell extracts were analyzed by anti-phosphoMad antibody for Dpp signals (upper panel) and by anti-Flag antibody for total Mad protein expression (lower panel). Dpp signaling was strongly blocked by Sog and Tsg, but this inhibition was reversed by the co-incubation with Tld. (B) Dpp signal (10^-10 M Dpp, 1/10 the amount used in A) was blocked by the same concentration of Sog as used in A, and this inhibition was partially reversed by Tld (lane 3). (C) The indicated combination of proteins was incubated as in A, and the collected supernatants were applied to a 4-12% NuPAGE gel and processed fragments were detected by western blotting using anti-Sog-CR1 and anti-Myc antibodies. Full-length Sog is barely detectable in Dpp/Sog/Tsg/Tld. Cleaved C-terminal fragments were detected by anti-Myc antibody, but with the exception of fragment (a) N terminal fragments are not detected by anti-Sog-CR1 antibody, suggesting that, in the presence of cells, these fragments are unstable (compare with Fig. 3A). (D) The supernatants of the indicated protein combinations from the experiment shown in B were analyzed for processing. Sog is partially cleaved by the incubation with Tld, and Dpp signals were partially restored. In all panels, arrows labeled a-g correspond to the fragments illustrated in Fig. 3B.