Figure 3: Screening for GEFs that regulate the Golgi pool of Cdc42.

(a) HEK293 cells were transfected with control or with the indicated siRNA. After 48 h, cells were transfected with the plasmid encoding for the Cdc42-EM-FRET probe. After a further 24 h the cells were fixed and mounted. FRET was measured as described in the Methods section and FRET values are displayed as fold of control and are averages from the measurements of at least 30 cells per condition. Red bars represent CI95 intervals (b) HeLa cells were transfected with plasmids encoding the indicated GEF. Twenty-four hours later, cells were lysed and the lysates were subjected to immunoprecipitation either with a GM130 antibody (+) or with protein G sepharose beads alone. ‘5% input’ indicates lanes where 5% of the total material that was used in the immunoprecipitation is loaded. The immunoprecipitated material was eluted and subjected to SDS–polyacrylamide gel electrophoresis followed by immunoblotting against the indicated proteins. (c) HeLa cells were plated on coverslips, then transfected with plasmids encoding the specified GEF. Twenty-four hours later, cells were fixed and immunostained for GM130 and Flag. Intensity plot corresponding to the region marked by a white arrow are shown on the right. Scale bar, 10 μm.