Figure 5.

Phosphorylated SNAP-23 resides in the cytosol. HeLa cells expressing syntaxin 4, SNAP-23, and SNAK were labeled for 3 h with [³²P]orthophosphate. After mechanical cell disruption, the postnuclear supernatant was separated into a membrane fraction and a cytosol fraction as described in the text. Triton X-100 was added to each fraction, immunoprecipitations were performed with the use of the indicated antiserum, and equivalent portions of the immunoprecipitates from each fraction were analyzed by SDS-PAGE and fluorography (upper panel). The immunoprecipitates were also probed by immunoblotting with a SNAP-23 antiserum to detect the total amount of SNAP-23 present in each sample (lower panel).