Figure 8.

Granuphilin interacts with Munc-18 in vitro and in living cells. (A) GST or GST-granuphilin 1–300 (GST-Gran) were immobilized on glutathione agarose beads and incubated with in vitro translated ³⁵S-labeled syntaxin-1, SNAP-25, VAMP-2, and Munc-18 (IVT). The proteins remaining associated with the affinity columns were separated by SDS-PAGE and visualized by autoradiography. (B) HIT-T15 cells were cotransfected with a fusion protein of GAL4 with Munc-18, a fusion protein of VP16 with the indicated mutants of granuphilin-b, and a firefly luciferase reporter gene. The luciferase activity produced in cells expressing the fusion proteins of GAL4 with Munc-18 and VP16 with wild-type (wt) granuphilin-b was set to 100%. The results are the mean ± SD of three independent experiments performed in duplicate.