Fig. 4. Region 2 of the Cyc pro-domain renders chimeric Sqt unstable, and targets the precursor for degradation. (A) Schematic representation of Cyc, Sqt and Sqt^proCyc2:Sqt. Hatched boxes indicate the leader sequences, the Cyc pro-domain is shown in pink, the Sqt pro-domain in dark blue, the Sqt mature domain in pale blue and the Cyc mature domain in mid-blue. (B,C) Analysis of induction of gsc (B) or ntl (C) in wild-type embryos injected at the one-cell stage with 0.5, 5 or 25 pg of RNA encoding Cyc, Sqt or Sqt^proCyc2:Sqt. Animal pole views of embryos at 50% epiboly showing endogenous gsc or ntl expression (I), and mild (II) or massive (III and IV) expansion of the gsc or ntl expression domains. Embryos were assessed and counted, and percentages for each class are shown in histograms. Scale bars: 100 µm. (D) 5 pg of RNA encoding Cyc, Sqt or Sqt^proCyc2:Sqt was injected into one cell at the 64- to 128-cell stage together with a lineage tracer (brown). Range of signaling was examined by in situ hybridization to detect ntl (blue, top panel) and gsc (blue-brown, bottom panel) expression. The Sqt^proCyc2:Sqt fusion behaves like Cyc, with its reduced ntl expression domain and the lack of gsc expression in most embryos (see Table S7 in the supplementary material). Scale bars: 100 µm. (E) Supernatants from HEK293T cells expressing Myc-tagged Sqt, Cyc and Sqt^proCyc2:Sqt proteins. Both unprocessed (46 kDa) and processed (17 kDa) forms of Sqt are detected. For Sqt^proCyc2:Sqt, the precursor is typically not detected and the processed Sqt (17 kDa) protein levels are reduced. The processed form of Cyc (16 kDa) is detected at very low levels. Supernatants from cells expressing pCS2Myc were used as negative control. GFP expression in the cell lysates was used to measure transfection efficiency. (F) Immunoblots to detect Myc-tagged Cyc (58 kDa), Cyc2 (55 kDa), Sqt (46 kDa), Sqt^proCyc2:Sqt (46 kDa), hNODAL (41 kDa) or Bmp2b (48 kDa) in extracts of cells grown in the presence or absence of the lysosomal inhibitor chloroquine. Cyc2 expression is not significantly altered by chloroquine treatment. By comparison, Cyc and the other proteins are stabilized by chloroquine. GFP was used as the transfection control and tubulin was used as the loading control. The histogram shows normalized protein expression levels relative to the GFP control, from three independent experiments (mean±s.e.m.). The asterisks (^*) indicate significant differences (P<0.05 as determined by paired Student's t-test) between cells not treated versus those treated with the lysosomal inhibitor for 24 hours. A representative gel from three independent experiments is shown.