Numb is a target of Lnx2a in the zebrafish pancreas. (A and B) Lnx2a is an E3 ubiquitin ligase that mediates the ubiquitination and degradation of Numb (16, 17). Myc-tagged WT Lnx2a and I51R and APAA mutants were co-transfected into 293T cells with T7-tagged Numb (A) and HA-tagged ubiquitin (B). After 36 h, immunoprecipitation and immuno blotting were performed with the indicated antibodies. (A) Numb interacts with WT and I51R mutant, but not with APAA mutant Lnx2a. In particular, co-immunoprecipitated Numb with WT Lnx2a was highly modified. (B) poly-ubiquitination of Numb was induced by WT Lnx2a but not by the I51R or APAA mutant. (C) Expression pattern of numb during early pancreas development. Transcripts of numb were detected in the whole pancreas, liver and intestine at 48 hpf, whereas numb-like was found in the brain but not in the endoderm. (D) Immune-staining with Numb antibody in Tg(ptf1a:EGFP) embryos reveals that Numb is expressed in the primary islet but not in ptf1a:EGFP+ ventral pancreas. At 48 hpf, Tg(ptf1a:EGFP) embryos were stained with anti- Insulin and anti-Numb antibodies, and confocal images in three axes are shown. (E) Immuno staining shows that exocrine pancreas defects in lnx2aΔ329 mutants and lnx2a-MO injected embryos are rescued by the knock-down of Numb. After numb-MO injection into WT siblings, lnx2aΔ329 mutants, and lnx2a-MO injected embryos, embryos were stained with anticarboxypeptidase A (α-cpa) and ductal cell-specific antibody 2F11, at 60 hpf. Stained embryos were analyzed by confocal laser scanning microscopy, and Z projections are shown. lnx2aΔ329 mutants and lnx2a-MO injected embryos showed defects in Cpa+ exocrine pancreas that could be rescued by knock-down of Numb. Li, Liver; In, Intestine; Pa, Pancreas; Pf, Pectoral fin; GB, Gall bladder; Scale bar, 50 µm (A and E) and 10 µm (B).