Figure 8.

SNAK enhances the expression of newly synthesized SNAP-23. (A) HeLa cells expressing SNAP-23 in the absence or presence of SNAK were labeled with [³⁵S]methionine for 15 min and chased in complete medium. After incubation for various times at 37°C, the cells were harvested, lysed in Triton X-100, immunoprecipitated with anti-SNAP-23 serum, and analyzed by SDS-PAGE and fluorography. The mobility of phosphorylated SNAP-23 (asterisk) was determined by analysis of ³²P-labeled SNAP-23 and [³⁵S]methionine-labeled SNAP-23 on the same SDS-PAGE gel. Note that in the presence of SNAK, phospho-SNAP-23 is observed after the pulse radiolabeling with [³⁵S]methionine. (B) The relative amount of [³⁵S]SNAP-23 precipitated with the SNAP-23 serum in the absence (□) or in the presence (▪) of SNAK was determined by phosphorimager analysis. The recovery of [³⁵S]SNAP-23 for each data point was expressed as a fraction of the amount of SNAP-23 recovered from cells coexpressing SNAP-23 and SNAK after the pulse radiolabeling (time 0). Each value represents the results from two independent experiments with SD.