Table 1.
Primers used for SYBR Green RT-qPCR.
Fig 1.
SNAP23 is expressed in human endothelial cells and murine tissues.
(A) Heat map of the gene expression values for SNAP homologs in 6 samples of human umbilical vein endothelial cells (HUVEC) by microarray. Relative expression values were normalized to that of GAPDH. (B) Relative expression of SNAP homologs in 3 individual donors of HUVEC as assayed by RT-qPCR. Expression was normalized as percentage of SNAP23 expression. Primers listed in Table 1. (C) ENCODE data on the UCSC genome browser depicting the expression of HUVEC SNAP homologs as assayed by RNA-seq. (D) SNAP23 is expressed in human brain microvascular endothelial cells (HBMEC), human aortic endothelial cells (HAEC), and HUVEC as measured by Western blot. (E) SNAP23 is expressed in multiple murine tissues as measured by qRT-PCR (n = 3 ± SD).
Fig 2.
Subcellular localization of SNAP homologs in endothelial cells.
Confocal microscopy was used to define the location of SNAP homologs (red) including SNAP23 (A), SNAP25 (B), SNAP29 (C), SNAP47 (D), VWF (green), and DNA (blue), and the images were merged. Only SNAP23 is localized to the plasma membrane.
Fig 3.
SNAP23 but not other SNAP homologs regulate VWF exocytosis.
(A) The effect of siRNA targeting individual SNAP homologs. HUVEC were transfected with siRNA against SNAP homologs or control siRNA, stimulated with media or histamine, and VWF release was measured by an ELISA. Knockdown of SNAP23 decrease stimulated VWF release, but knockdown of other SNAP homologs has no effect on VWF release (n = 4–6 ± S.D.). (B) Comparison of the role of SNAP23 and SNAP29 in mediating exocytosis. The effect of SNAP23 and SNAP29 in endothelial exocytosis after RNAi was measured as above. Knockdown of SNAP23 decreased exocytosis, but knockdown of SNAP29 had no effect (n = 14–16 ± S.D.).
Fig 4.
SNAP23 is important for endothelial exocytosis.
(A) siRNA against SNAP23 knocks down SNAP23 protein levels as measured by Western blot. The siRNA against SNAP23 has no effect on the expression of other SNARE proteins including STX4, VAMP3, and VAMP8. GAPDH was used as loading control. (B) SNAP23 knockdown does not affect VWF expression in HDMVEC or HUVEC. Total VWF content was measured in total cell lysate by an ELISA in control siRNA and siSNAP23 treated cells (n = 6; NS, non-significant). (C) SNAP23 knockdown decreases endothelial exocytosis. HDMVEC and HUVEC were treated with siControl or siSNAP23, stimulated with serum-free medium only (resting), or 10 μM histamine, or 1 U/ml thrombin, or 10 μM Ca2+ ionophore A23187 for 30 min; and then VWF released into the media was measured by an ELISA (n = 4–7; * P < 0.05 vs. siControl; NS, non-significant vs. siControl). Data are represented as mean ± SD.
Fig 5.
Subcellular localization of SNAP23 in endothelial cells.
(A) Subcellular localization of SNAP23 in sub-confluent (upper panel) and confluent (lower panel) endothelial cells. Immunofluorescent staining was performed on HUVEC with antibodies against SNAP23 (red) and VWF (green), DNA was stained with DAPI (blue), and the cells were imaged by confocal microscopy (objective 60× oil, scale bar = 40 μm, confocal z resolution = 0.32 μm). (B) Western blot analysis of cell fractions from sub-confluent and confluent HUVEC using markers for membrane (caveolin-1 or CAV1) and cytosol (GAPDH). These fractions were also probed for SNAP23. Whole cell lysates were used for total protein. SNAP23 expression is decreased from cytosol fraction when cells are confluent.
Fig 6.
SNAP23 interacts with endothelial exocytic machinery.
(A) SNAP23 co-sediments with STX4, VAMP3 and VAMP8 as analyzed by sucrose density gradient fractionation. HUVEC lysates were ultracentrifuged through a 5%–40% discontinuous sucrose gradient, and then the gradient was aliquoted into 17 fractions and analyzed by SDS-PAGE (T, total proteins in the lysate; P, pellet after fractionation). β-actin was used as control for fraction separation. Representative of 3 separate experiments. (B) SNAP23 co-precipitates with STX4, VAMP3 and VAMP8. HUVEC were stimulated with serum-free medium only (Rest), or 10 μM histamine, or 10 μM Ca2+ ionophore A23187 for 30 min; and cell lysates were immunoprecipitated with antibody to SNAP23 or isotype IgG. The precipitants were probed with antibody to STX4, VAMP3 and VAMP8. Input represents 5% total protein. Representative of 3 similar experiments. SNAP23 co-precipitates with STX4, VAMP3, and VAMP8.