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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).
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