DEV0197944 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Amino acid alignment of hNodal and the zebrafish Nodal-related proteins Cyc, Sqt and Southpaw. The cleavage sites of hNODAL, Cyc, Sqt and Southpaw are boxed in grey, and the seven characteristic cysteine residues in the mature domain of TGFβ proteins are shown in red. The predicted glycosylation sites in Cyc are highlighted in green. Region 1-12 of the Cyc pro-domain and the corresponding regions in the other Nodal-related proteins are underlined. A putative lysosomal targeting region in region 2 of Cyc is highlighted in yellow. The red box marks the mutated Arg residues conserved between hNodal and Cyc, but not present in the other two zebrafish Nodal-related proteins.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Nodal-related proteins are stabilized by 24-hour treatments with the proteasome inhibitor MG132. Immunoblots to detect Myc-tagged Cyc (∼58 kDa), Cyc 2 (∼55 kDa), Sqt (∼46 kDa), SqtproCyc2:Sqt (∼46 kDa), hNodal (∼41 kDa) or BMP2b (∼48 kDa) in extracts of cells grown in the presence or absence of the proteasome inhibitor MG132 for 24 hours show that all proteins are stabilized by treatment with MG132. Expression of GFP transfection control and Tubulin loading controls are shown. The graph shows normalized protein expression levels from three independent experiments, relative to the GFP control (mean±s.e.). The asterisk denotes significant difference (P<0.05 as determined by paired Student's t-test) between cells that were not treated versus those treated with the proteasome inhibitor for 24 hours.

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