Figure 4.

SNAP-23 in t-SNARE complexes is not phosphorylated. (A) HeLa cells expressing syntaxin 4, SNAP-23, and SNAK were labeled with [³²P]orthophosphate, lysed in Triton X-100, subjected to immunoprecipitation with the indicated antiserum, and analyzed by SDS-PAGE and fluorography. (B) HeLa cells were transfected with a constant amount of SNAP-23 and SNAK cDNA (2 μg each) and increasing amounts of syntaxin 4 cDNA (0, 0.1, 0.5, or 2 μg) with the use of Lipofectamine. The cells were then labeled with [³²P]orthophosphate, lysed, immunoprecipitated with the indicated antiserum, and directly analyzed by SDS-PAGE and fluorography (upper panel). The immunoprecipitates were also probed by immunoblotting with a SNAP-23 antiserum to detect total SNAP-23 and t-SNARE–associated SNAP-23 (lower panels).