Fig. 5.

Phosphorylated syntaxin 1 is enriched in complexes with SNAP-25. A, Stimulation of acute rat brain slices failed to alter the state of serine-14-phosphorylation on syntaxin1A/B. Acute rat brain slices were prepared from adult animals and subjected to no stimulation (NS), 1 hr in 56 mm potassium (K+), 20 min in 50 μm 4-amino-pyridine (4AP), 1 hr in 10 μm phorbol 12,13-dibutyrate (PDBu), or 1 hr in 30 μm sphingosine (SPHI). The slices were homogenized, and the homogenate was extracted with Triton X-100. Total protein (50 μg) of each lysate was resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with αPsyn.B, In vitro-phosphorylated syn1A11 readily assembles with VAMP2Δ1–24, SN25C, and SN25N to form an SDS-resistant/boiling-sensitive core complex. Syn1A11 was in vitro-phosphorylated with purified CKII and then mixed with a twofold molar excess of VAMP2Δ1–24, SN25C, and SN25N. After a 6 hr incubation, unboiled (lane 1) or boiled (lane 2) samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with αPsyn. Under unboiled conditions (lane 1), phosphorylated syn1A11 was found primarily in a ∼50 kDa SDS-resistant complex (arrow) with little protein left in the monomeric form (asterisk). After boiling (lane 2), the SDS-resistant complex disassembled, and the phosphorylated syn1A11 was detected exclusively in the monomeric form. C, Phosphorylated syntaxin 1A/B coimmunoprecipitates with SNAP-25. Rat brain was homogenized, and the postnuclear fraction was extracted with Triton X-100. The lysate was incubated with a αSNAP-25 antibody followed by protein-G coupled to Sepharose beads. Total protein (25 μg) from the starting material (ST) and one-fifth of the total immunoprecipitated material (IP) were loaded on duplicate and resolved by SDS-PAGE, transferred to nitrocellulose membranes, and finally probed to detect the presence of phosphorylated syntaxin 1A/B (αPsyn), total syntaxin (αHPC-1), SNAP-25, andVAMP2.