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 Cyc2 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.