Syntaxin 4 in human platelets and its interaction with PSP. (A) Platelet lysates treated with Triton X-100 (1%) were subjected to SDS-PAGE on 15% gels and electroblotted to membranes. The blots were incubated with (lane 1) preimmune serum at 1:1,000, (lane 2) anti-syntaxin 4 antibody at 1:1,000, (lane 3) anti-syntaxin 4 antibody absorbed with recombinant glutathione-S transferase–syntaxin 4 at 1:1000, and (lane 4) anti-syntaxin 4 antibody absorbed with recombinant glutathione-S transferase at 1:1,000. Relative positions of molecular mass standards are indicated in kilodaltons. (B) Platelet cytosolic (C) or particulate (P) fractions (see above) were subjected to SDS-PAGE on 7.5% gels and immunoblotted with anti-syntaxin 4 antibody (1:1,000) as described above. The arrow indicates syntaxin-4 immunoreactivity. (C) Interaction of syntaxin 4 with PSP. r-PSP (left panel, 10 μg/300 μL) or human platelet lysate (right panel, 300 μL) was incubated with r-syntaxin or r-SNAP-25 coupled to glutathione sepharose beads overnight at 4°C. After a wash, protein bound to the beads was eluted with sample buffer and subjected to SDS-PAGE followed by immunoblotting with the anti-PSP peptide antibody (1:1,000). The arrow indicates the PSP-immunoreactive band. (D) Competition between r-SNAP-25 and r-PSP for binding to r-syntaxin 4. Various amounts of r-SNAP-25 (0 to 425 μg/mL, final) were mixed with¹²⁵I-r-syntaxin 4 (∼100,000 cpm) and 50 μL was added to microtiter plate wells coated with r-PSP (50 μL, 0.15 μg/mL) in duplicate. After 1 hour, the wells were washed and the amount of bound¹²⁵I-r-syntaxin 4 was determined by γ scintillation counting. The percentage of binding inhibition was determined by computing the fractional binding of ¹²⁵I-r-syntaxin 4 to PSP in the presence of various amounts of r-SNAP-25, in comparison with the binding in the absence of an inhibitor, after correcting for nonspecific binding. (E) PKC-phosphorylation inhibits the binding of PSP to syntaxin 4. Microtiter plates coated with baculovirus expressed PSP or no PSP (background) were incubated with r-syntaxin 4 in the presence of equal amounts of various inhibitors: no PSP (none), PSP, mock-phosphorylated PSP, and PKC-phosphorylated PSP. To increase the sensitivity of the assay, the amount of PSP added as competitor was calibrated by preliminary assays to produce approximately 50% inhibition of r-syntaxin 4 binding. The amount of r-syntaxin 4 binding to the PSP-coated wells was determined by measuring the binding of anti-syntaxin 4 antibodies as detected by ¹²⁵I-protein A.