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