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Fig. 2. Specific regions within the Cyc pro-domain regulate its expression
levels and activity. (A) Schematic representation of the Cyc
precursor. The Cyc leader sequence is shown by the hatched box, the pro-domain
in pink and the mature domain in blue. Regions 1-12 are 30 amino acid
stretches in the pro-domain. (B) Analysis of induction of gsc
in wild-type embryos injected with 1.25 pg aliquots of RNA encoding Cyc with
deletions in regions 1-12 of the pro-domain. Some deletions, such as
Cyc
2 and Cyc9, have stronger gsc-inducing activity than
wild-type Cyc, whereas some have either no detectable activity (Cyc4) or
reduced activity. Scale bar: 100 µm. (C) Expression of FLAG
epitope-tagged versions of the various Cyc proteins. Immunoblots on extracts
of HEK293T cells expressing FLAG-tagged Cyc proteins show more Cyc1 and
Cyc2 protein than Cyc or the other deletions. The protein samples were
loaded on two gels (lanes 1-7 and 8-14, respectively), and electrophoresed in
parallel. Western blots of the input proteins are shown, detected with
antibodies towards the FLAG epitope for the Cyc proteins, 
-GFP for
transfection control and -Tubulin for loading controls. The graph shows
normalized protein expression levels relative to the GFP control, from three
independent experiments (mean±s.e.m.). Asterisks indicate significant
differences; P<0.05 by paired Student's t test. A
representative blot is shown.
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