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Fig. 5. Downregulation of Syt I or Syt IX alone has no effect on stimulated exocytosis. (A) During isolation of stable cell lines lacking Syt I and Syt IX, clones were isolated in which Syt I was fully downregulated but Syt IX was expressed at wild-type levels (Syt I-null cells). pSHAG-Syt IX downregulated Syt IX without affecting Syt I expression (Syt IX-null cells). Immunoblot analysis was used to analyze Syt I and Syt IX. (B) Wild-type, Syt I-null and Syt IX-null cells were transfected with a plasmid expressing hGH and intact cell secretion assays were conducted. Ca²⁺-dependent hGH release was unaffected by the downregulation of either Syt I or Syt IX. (C) Wild-type, Syt I-null and Syt IX-null cells, transfected with a plasmid expressing ANF-EGFP, were stimulated in depolarization medium and TIRF images were acquired at 0.25-second intervals. The data were analyzed as in Fig. 4 and plotted as the sum of the events for ten wild-type, ten Syt I-null, ten Syt IX-null and ten Syt I/IX-null cells. DCV exocytosis occurred in the Syt I-null or Syt IX-null cells. (D) The sum of the average number of fusion events per cell over time (mean ± s.d.) for wild-type (n=30), Syt I-null (n=15) and Syt IX-null cells (n=10). values for fusion in wild-type, Syt I-null and Syt IX-null cells were 32.1 seconds, 36.0 seconds and 17.9 seconds, respectively. Differences between Syt IX-null and wild-type or Syt I-null were significant (P<0.001).

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