**3.4.4 Enzymatic cell fusion assay**

The day before transfection, 1.2 × 106 COS-7 cells are seeded in each 100-mm cell culture dish, and 2 × 105 COS-7 cells are seeded in each well of 6-well plates. For v-cells, 5 g each of flipped v-SNARE plasmid are cotransfected with 5 µg of pTet-Off into the cells in each 100 mm culture dish. Control cells are cotransfected with empty vector pcDNA3.1(+) and pTet-Off. For t-cells, 1 µg each of flipped syntaxins 1 or 4, SNAP-25 and pBI-G are cotransfected into the cells in each well of the 6-well plates. There are putative N-glycosylation motifs (Asn-X-Ser/Thr) in VAMPs. Our previous studies (Hu et al., 2003; Hu et al., 2007) showed that N-glycosylation disrupts the function of flipped SNAREs, and that treatment of tunicamycin, an antibiotic that inhibits N-glycosylation (Elbein, 1984), restores the fusion activities of flipped SNAREs. To prevent N-glycosylation of VAMPs 1, 4, 5, 7 and 8, v-cells expressing these VAMP proteins and control cells are incubated in cell culture medium containing 10 g/ml of tunicamycin during transfection. Since we have mutated the putative N-glycosylation sites in flipped VAMP2 (Hu et al., 2003) and VAMP3 proteins (Hu et al., 2007), v-cells that express VAMPs 2 or 3 are not treated with tunicamycin. Since flipped syntaxins 1 or 4, SNAP-25 proteins are not N-glycosylated (Hu et al., 2003; Hu et al., 2007), the t-cells are not treated with tunicamycin.

Twenty-four hours after transfection, the v-cells are detached from the culture dishes with Enzyme-free Cell Dissociation Buffer, and added (4.8 × 105 cells) to each well already containing the t-cells. After 6, 12 or 24 hours at 37C in 5% CO2, the expression of βgalactosidase is measured using the β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer according to the manufacturer's instructions (Promega). The cells are washed twice with PBS, and then lysed in Reporter Lysis Buffer. Cell lysates are mixed with equal volume of Assay 2× Buffer. As a blank control, Reporter Lysis Buffer is mixed with Assay 2× Buffer. After 90 minutes, the colorimetric reaction is stopped by adding 1 M sodium carbonate, and absorbance at 420 nm is measured using a HITACHI 100-40 spectrophotometer.
