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. 2004 May 24;101(22):8313–8318. doi: 10.1073/pnas.0306709101

Fig. 3.

Fig. 3.

Interaction of Noc2 and Rab3, Rab3 effector expressions, and rescue of impaired insulin secretion in Noc2-/- by Noc2 transgene. (a) Interaction of Noc2 and various Rab3 isoforms. Lysate from COS-1 cells transfected with Flag-tagged Rab3 isoforms and Rab5 were evaluated for binding to GST-Noc2 in the presence of GDP-βS or GTP-γS. Rab3 isoforms and Rab5 and GST-Noc2 were detected by immunoblotting with anti-Flag antibody or IgG-purified antibody against rat Noc2. GST-Noc2 binds to all isoforms of Rab3 in the presence of GTP-γS, but does not bind to Rab5. (b) Rab3 effector mRNA expressions. For amplification of rabphilin3, brain cDNA was used as a positive control of PCR, because rabphilin3 is expressed in brain but not in pancreatic islets (41). α-tubulin, a housekeeping gene, was used as a positive control for cDNA synthesis from pancreatic islets. (c) Interaction of Noc2wt or Noc2AAA and GTP forms of various Rab3 isoforms. Noc2AAA was generated by replacing WFY (residues 154–156) with alanines, as described (25). Lysate from COS-1 cells transfected with Flag-tagged Rab3 isoforms was evaluated for binding to GST-Noc2wt or GST-Noc2AAA in the presence of GTP-γS. Mutant Noc2 does not bind to any of the isoforms of Rab3. (c) Rescue of impaired insulin secretion in Noc2-/- mice by Noc2 transgene. Pancreatic islets of Noc2-/- mice were infected with recombinant adenoviruses carrying LacZ, Noc2wt, or Noc2AAA cDNA at the same titers (multiplicity of infection, 100) for 48 h after isolation. Ad-LacZ, Ad-Noc2wt, and Ad-Noc2AAA indicate adenovirus carrying LacZ, Noc2wt, and Noc2AAA, respectively. Defective Ca2+-triggered insulin secretion in Noc2-/- mice is completely restored by Noc2wt gene transfer (Ad-LacZ, 0.69 ± 0.07 ng per islet per 30 min; Ad-Noc2wt, 1.20 ± 0.11 ng per islet for 30 min, n = 9; *, P < 0.01), but the mutant Noc2 has no effect (Ad-Noc2AAA: 0.65 ± 0.11 ng per islet per 30 min, n = 9). Open columns, Noc2+/+ mice; filled columns, Noc2-/- mice. Values are means ± SEM.