Open Access Research article

The putative Notch ligand HyJagged is a transmembrane protein present in all cell types of adult Hydra and upregulated at the boundary between bud and parent

Andrea Prexl1, Sandra Münder1, Bernhard Loy2, Elisabeth Kremmer3, Susanne Tischer1 and Angelika Böttger1*

Author Affiliations

1 Department of Biology 2, Ludwig-Maximilians-Universität München, Munich, Germany

2 Institute for Neuroscience, Innsbruck Medical University, Innsbruck, Austria

3 Institute for Molecular Immunology (IMI), Helmholtz Zentrum München, Munich, Germany

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BMC Cell Biology 2011, 12:38  doi:10.1186/1471-2121-12-38

Published: 7 September 2011

Additional files

Additional file 1:

Testing of rabbit anti-JAG-IC peptide antibody. (A-C) Western Blot after SDS-PAGE with bacterial lysates from E.coli expressing HyJagged-ICD (10 kDa) from pRSET (lanes labelled R JAG-IC 250 ng) and with vesicle fraction from hydra homogenates of 125 animals (lanes labelled V 125 a) probed with anti-JAG-IC antibody (A); Control with rabbit pre-immune serum (B); (C) Control Western blot probed with an unrelated rabbit antibody. This antibody had been raised against a peptide derived from Hydra HES and recognises HyHES (25 kDa) in bacterial lysates from E.coli expressing HyHES from pREST (lane RHyHES 50 ng). (D-G) HEK293T cells expressing HyJagged-GFP from pcDNA3; (D) DNA staining with TO-PRO3, (E) HyJagged-GFP, (F) anti-JAG-IC staining, (G) merged images in false colours: DNA (blue), HyJagged-GFP (green), anti-JAG-IC staining (red); Confocal sections; scale bar: 5 μm.

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Additional file 2:

Characterization of anti-ZIC7A12 antibody. (A, B) Western blot after SDS-PAGE of bacterial lysates expressing HyZic from pRSET (lanes recomb. HyZic 50 ng and 10 ng) and lysates from hydra cells (lanes Hydra Ly) probed with monoclonal rat antibody anti-ZIC7A12 (A). Control with a rat IgG that had been raised against CnASH (B). This antibody recognises a band below 43 kDa in hydra lysates. The anti-ZIC7A12 recognises a band at 46 kDa, which corresponds to the predicted size for HyZic. (C-F) Single hydra epithelial cell ectopically expressing HyZic-GFP fusion protein from HotG after transfection of the animals with particle gun. (C) DNA staining with TO-PRO3, (D) HyZic-GFP, (E) anti-ZIC7A12 staining, (F) merged images in false colours: DNA (blue), HyZic-GFP (green), anti-ZIC7A12 staining (red); Confocal sections; scale bar: 2 μm.

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Additional file 3:

Characterisation of anti-Notch antibody. (A-C) Western Blot after SDS-PAGE of lysates from E.coli expressing myc tagged HvNΔE (49 kDa) from pRSET probed with anti-Notch antibody (A), probed with chicken preimmune serum (B), probed with anti-myc antibody (C); arrows indicate HvNΔE and two apparent degradation products; (D-G) HEK293T cells expressing HvNotch-GFP from pcDNA3. (D) DNA staining with TO-PRO3, (E) HvNotch-GFP, (F) anti-Notch antibody staining (G) merged images in false colours: DNA (blue), HvNotch-GFP (green), anti-Notch staining (red); Confocal sections; scale bar: 5 μm.

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