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Fig. 2. shRNA knockdown of Syt I and IX abolishes regulated exocytosis. (A) Nucleotide sequence alignment of Syt I and Syt IX in the part of the ORF targeted by shRNA. Predicted structure of the shRNA transcript (Syt I/IX). (B) Clones stably expressing pSHAG-Syt I/IX (Syt I/IX-null) were isolated and an immunoblot of a representative clone is shown using Syt-I-specific and Syt-IX-specific antibodies. The Syt I/IX-null cells were further characterized with Syt-IV-specific and Syt-VII-specific antibodies. SNAP-25 staining was used as a loading control for both immunoblots. (C) Wild-type and Syt I/IX-null cells were transfected with a plasmid encoding hGH, and intact cell secretion assays were conducted. Ca²⁺-dependent hGH secretion was determined as the amount of hGH secreted in 20 minutes at 37°C, stimulated by depolarization medium (K⁺-stimulated) minus the amount of hGH secreted in basal medium (basal). Values represent the mean of triplicate determinations, the standard diviation (s.d.) is indicated. Ca²⁺-dependent DCV exocytosis was similarly abolished in three Syt I/IX-null cell lines and in transient transfections with the Syt I/IX-shRNA plasmid. (D) hGH secretion assays in intact cells were conducted at the indicated time points. Ca²⁺-dependent hGH secretion (K-stimulated minus basal) was plotted as percent hGH release (total hGH secreted divided by total hGH secreted plus total hGH remaining in the cells x100). The results shown are representative of three independent experiments and similar results were obtained for three Syt I/IX-null cell lines. (E) Time course for basal and K⁺-stimulated hGH release of wild-type and Syt I/IX-null cells. In Syt I/IX-null cells, release of hGH was the same in the absence or presence of a stimulus, and was significantly reduced compared with hGH release in K⁺-stimulated wild-type cells. Results are representative of three independent experiments and were similar for three Syt I/IX-null cell lines. (F) Immunocytochemistry of CgB (DCV marker) and transiently transfected hGH in wild-type and Syt I/IX-null cells. hGH is targeted to DCVs in wild-type and Syt I/IX-null cells. The cellular distribution of DCVs was unaffected by the downregulation of Syt I and Syt IX. Bar, 10 µm.

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