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Your Data: Use of pSilencer 1.0-U6 siRNA Expression Vector in the Analysis of Synaptotagmin Location and Function

ncreatic islets. Confocal microscopy showed that SytV and Syt IX co-localize with insulin-containing secretory granules which was confirmed by using a continuous sucrose density gradient. Interestingly, studies performed in primary pancreatic endocrine cells revealed that Syt V is present in glucagon-containing islet alpha-cells but is not expressed in β-cells whereas Syt IX is associated with insulin granules in β-cells.

To investigate the function of Syt V and Syt IX, the Syt isoforms were over-expressed or their levels reduced using RNA interference. Both proteins were produced at similar levels but the anti-Syt IX antibody recognised a smaller and a larger form of Syt IX. The two forms of Syt IX may be due to post-translational modification such as O-glycosylation and palmitoylation. Transient over-expression of Syt V and Syt IX did not alter exocytosis in INS-1E cells indicating that both proteins do not interfere with the stoichiometry of SNARE proteins whose over-expression has been shown to inhibit insulin exocytosis (Nagamatsu et al., 1999).

To investigate the hormone release, the pSilencer SytV and Syt IX siRNA expression plasmids were each transiently cotransfected into INS-1E cells with a plasmid encoding the human growth hormone hGH. hGH is targeted to insulin-containing granules and can be used to monitor exocytosis as an insulin substitute in a subpopulation of transfected cells (Iezzi
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