Fig. 3. Sog processing is enhanced by a Dpp/Gbb heterodimer. (A) Western blot showing a representative sample of ligand produced by cells expressing either Dpp–HA alone (lane 1), Gbb (lane 3) or co-transfected Dpp-HA and Gbb (lane 2). The immunoblots were simultaneously probed for Dpp-HA with anti-HA 12CA5 antibody and Gbb with rat anti-Gbb C terminus antibody. The blots were scanned with Odyssey Infrared Imaging System. (B) Co-expression of Dpp-HA and Gbb-Flag leads to heterodimer formation. The top panel is probed for the presence of Dpp-HA, while the bottom panel is probed for Gbb-Flag using the anti Flag M2 antibody. Lanes 1-4 show the input levels of ligands present in the starting samples. Lanes 5-8 are immunoprecipitations with the anti-HA antibody. Lanes 9-12 show immunoprecipitated material using the anti-Flag antibody. The red arrows indicate co-immunoprecipitation of Dpp and Gbb which is only seen in the samples (lanes 7 and 11) where the two ligands where co-transfected into the same S2 cells and not in samples where homodimers where mixed together (lanes 8 and 12). The 25 kDa band (black arrowhead) present in all anti-Flag M2 immunoreactions is due to the mouse IgG light chain detached from the beads under our experimental conditions. (C) Sog processing by Tld and Tlr (16 hours at 25°C) in the presence of Dpp or Gbb homodimers was compared with the processing in the presence of Dpp and Gbb heterodimers (Dpp/Gbb). Equivalent amounts of Bmp ligands estimated by their immunoreactivity (A) were included in the processing reactions as indicated. (D) Percentage of remaining full-length Sog relative to the no-enzyme containing processing reaction (lane 1) was quantified using the Odyssey Infrared Imaging System (see Materials and methods).