Figure 4: The effect of GM130 on Cdc42 is dependent on RasGRF.

(a) HeLa cells were lysed and the lysate was 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. (b) Quantification of cells with RasGRF at the Golgi. (c) Immunofluorescence staining in HeLa cells for RasGRF and GM130. Images were acquired using a confocal laser-scanning microscope. The magnifications correspond to the areas inside the white squares. Scale bar, 25 μm. An intensity plot corresponding to the region marked by a white arrow is shown. (d) CHL cells transfected with indicated siRNAs were lysed and GST-PAK1 was added to the lysate followed by pull down with GSH-sepharose. The eluate was subjected to immunoblotting against Cdc42 (Cdc42-GTP). Five per cent of the original lysate was immunoblotted against Cdc42 (Tot-Cdc42) and GM130. Bar graph represents a quantification of GTP-Cdc42 normalized to total Cdc42. Results are presented as mean±s.d. from three experiments. (e) Quantitative RT–PCR of RasGRF2 HeLa and CHL cells. (f) Confluent monolayer of cells transfected with the indicated siRNA was wound and cells were allowed to polarize for 6 h followed by fixation and staining of Giantin to label the Golgi. Golgis were counted as oriented towards the wound if their major mass was located in a 120° angle facing the wound. The red line shows the average orientation of the Golgi immediately after wounding. Results are presented as mean±s.d. from three experiments. (g) Caco-2 cells transfected with indicated siRNAs were grown in Matrigel for 4 days. Afterwards, cysts were stained for GM130 using immunofluorescence and for F-actin using phalloidin. Cysts with a single lumen were counted as normal, while cysts with multiple or no lumen were counted as aberrant. The percentage of normal and aberrant cysts in each condition was counted from three experiments which are displayed in the bar graph in the lower panel. Results are presented as mean±s.d. Scale bar, 7.5 μm. Statistics in F&G: analysis of variance with Newman–Keuls multiple comparison test (*P<0.05; **P<0.01).