Fig. 5. Pro-peptide processing activates Alp in a cell-based signaling assay. (A) Signaling activities of various ligands -/+ Tld were tested using S2 cells expressing Flag-Mad (left panels) or Flag-Smad2 (right panels) and western analysis of the cell extracts. The signals were analyzed using primary antibodies against Mad1-P and Smad2-P (P-Mad1 and P-Smad2, respectively) and an anti-Flag antibody, and anti-rabbit IRDye 800 and anti-mouse IRDye 700 secondary antibodies, followed by scanning and quantification by the Odyssey Infrared Imaging System. (B) Ligands +/-enzymes were analyzed using anti-V5 antibody for Daw pro-peptide detection (arrow) and anti-HA for enzymes detection (parenthesis). (C,E) Dose-dependence of Daw activities +/-Tld or Tlr in signaling assays. S2 cells transfected with Flag-Smad2 were incubated with 0, 25 µl, 50 µl, 100 µl and 150 µl of the conditioned supernatants shown in B, lanes 1, 2 and 4 (C), or 5, 6 and 8 (E). (D,F) Quantification of Tld and Tlr effects on Daw activities. The signals are normalized as the relative ratio Smad2-P/Flag and represented as a function of ligand amount.