FIGURE 4. EGFP fluorescence measurements after induction of GP64-mediated fusion at varying pH values.
Sf9 cells that were transfected with pEGFP-92lacO were mixed with LacR-IE1/GP64 expressing cells, then exposed for 20 min to Graces medium at pH values ranging from pH 5.0 to pH 6.0. Cells were then returned to pH 6.0 and incubated for 24 h. EGFP fluorescence was examined directly by fluorescence microscopy (Panel A) and by fluorescence measurements of cell lysates (Panel B). Numbers in panel A represent the pH values used to trigger membrane fusion. In panel B, pH values used to trigger membrane fusion are indicated on the X-axis and arbitrary fluorescence measurements are indicated on the Y-axis. Various cell treatments are indicated by symbols: Open circles (solid line) represent a group of cells expressing LacR-IE1 and GP64 mixed with a second group of cells containing reporter plasmid pEGFP-92lacO. Positive and negative control experiments are indicated by open and closed triangles, respectively. Open triangles (broad dashed line; positive control) represent a mixture of cells containing one group that was co-transfected with the activator protein (LacR-IE1) and the responsive EGFP reporter plasmid (pEGFP-92lacO) as described in Fig. 3C. Closed triangles (short dashed line) represent a mixture of cells containing one group of cells transfected with the responsive EGFP reporter plasmid, and another group that expressed LacR-IE1 (but no GP64) and were treated as described in Fig. 3B. Error bars represent standard deviations derived from 3 replicate wells of each treatment. The white bar represents 200 μM.