Supplemental Figure 1
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Fig. 1. Maternal β-catenin-2
protein levels are reduced in early ichabod embryos. (A-D)
Western blots of extracts of wild-type
(A,C) and ichabod (B,D) embryos at 100% (A,B) and 30% (C,D)
epiboly, injected with different combinations of MOs (indicated above the
lanes), were immunostained for b-catenin
(using a pan-b-catenin first antibody –
rabbit anti b-catenin, C2206, Sigma)
and for b-tubulin (as a normalizing
control, mouse monoclonal anti b-tubulin
– T0198, Sigma). The bound antibodies were detected with peroxidase-conjugated
donkey anti-rabbit IgG (NA934V, Amersham) and peroxidase-conjugated sheep
anti-mouse IgG (NA931V, Amersham), and protein bands were visualized with the
ECL Plus Western Blotting Detection System (Amersham). To keep the levels of MOs
the same in all cases, we injected either MO2 + MO1mis (to
specifically inhibit b-catenin-2
translation), MO2mis + MO1 (to specifically inhibit b-catenin-1 translation), MO2 + MO1 (to
inhibit both b-catenins) or the two
mismatched MOs (negative control). All MOs were injected at a concentration of
3 mM. (E,F) Results of densitometric scans of 30% (E) and 100%
(F) epiboly embryos, with relative b-catenin
protein levels indicated by blue bars for wild-type embryos and by green bars
for ichabod embryos. For each type of
embryo (e.g. 100% wild type, 100% ichabod, etc.), the values for the three specific MO
treatments have been normalized with respect to the band density value obtained
from embryos injected with the two control morpholinos (set to 1.0). (Band
densities were determined using BioRad Gel Doc XR System with Quantity One
software.) A table with the relative band densities is shown at the bottom.
Injection of MO2 or MO1 each reduces the level of b-catenin in 100% epiboly wild-type and ichabod
embryos, and in 30% wild-type embryos. In
these cases, co-injection with MO1 and MO2 further decreases the level of b-catenin. However, at 30% epiboly in ichabod embryos, only MO1 caused a decrease in b-catenin level (C, lane 4). MO2 did not
decrease the amount of the protein (C, lane 3) and a combination of the two MOs
was no more effective than MO1 alone (C, lane 5). We conclude that in contrast
to wild-type embryos at both stages and ichabod embryos at 100% epiboly, in which both b-catenins are present, ichabod embryos at 30% epiboly have very little, if any, b-catenin-2, although they do contain b-catenin-1. The failure to completely
eliminate b-catenin protein in the
embryos by treating with MOs could be due to the failure of the specific MOs to
eliminate all translation or to a contribution of pre-existing maternal
protein.
