Gα12 interaction with α-SNAP. (A) Design of N-terminal Gα12^QL substitution mutants with sequence Asn-Ala-Ala-Ile-Arg-Ser (NAAIRS). The ΔN10-15 Gα12^QL construct was generated by replacing 6 consecutive residues (aa 10-15) with NAAIRS; the ΔN4-9 Gα12^QL construct was generated by replacing aa 4-9 with NAAIRS. (B) Coimmunoprecipitation of Myc-tagged constitutively active Gα12^Q229L mutant expressed in HEK 293A cells with endogenous α-SNAP. Myc tagged Gα12^QL mutants³³  were expressed in HEK 293A cells, and their interaction with GST-αSNAP was analyzed by pull-down assay. Constitutively active Gα12-ΔN4-9 was observed to bind αSNAP similar to full-length Gα12^QL, whereas Myc-ΔN10-15 mutant bound much less to α-SNAP. (C) Densitometry values (mean ± SEM; n = 3 independent experiments). *P < .05 vs full length Gα12^QL. (D) Confocal images of functional dominant-negative effect of GFP-tagged-SBD (aaN10-15, the putative αSNAP binding domain) on vWF secretion in HUVECs. Twenty-four hours after transfection, cells were serum-starved for 4 hours, washed, incubated with 25 nM thrombin for 15 minutes, fixed, and stained for vWF (red). (E) Each symbol represents the number of WPBs per individual cell. GFP-tagged SBD functioned as a competitive inhibitor of basal and thrombin-induced vWF exocytosis from WPBs. *P < .05 vs unstimulated empty GFP vector transfected cells; n = 7-15 cells/group; red line represents median value.