Figure 8: IC87114-induced vesicle translocation is dependent on Cdc42.

(a) Bovine chromaffin cells expressing NPY–Cherry alone (control) or co-expressing either wild-type Cdc42 or the dominant negative Cdc42(T17N)–GFP (Cdc42DN) construct with NPY–Cherry were visualized by TIRF microscopy. Cells were imaged at 1 frame per second before and after addition of IC87114 (1 μM). The changes in fluorescence intensity of NPY–Cherry are displayed in pseudocolour. The images shown are taken from before the IC87114 treatment and at the peak of its effect. (b) The time-course variations of NPY–Cherry in the indicated conditions are expressed as percentage of the initial intensity. (c) Quantification of the peak fluorescence intensity increases (% of initial) is expressed as mean±s.e.m. (n=3–5 cells per condition). Scale bar, 10 μm. (d) Bovine chromaffin cells treated with DMSO (control) or 1 μM IC87114 (20 min) were lysed and assayed for activated Cdc42 (GTP–Cdc42) by western blotting. Control chromaffin cells were incubated with GDP (negative control) or GTPγS (positive control) and assayed. Bar graph shows the percentage of activated Cdc42 in control and IC87114-treated cells (n=3 independent experiments). Data are expressed as mean±s.e.m. Student's t-test *P<0.05.