Fig. 9. Effects of Rab18 mutants and knockdowns on rates of brefeldin-A-induced ER-Golgi fusion. (A) Frames taken from a representative time-lapse imaging sequence showing effects of brefeldin A (5 µg/ml)-induced `blinkout' in COS7 cells expressing Galtase-YFP. (B) Golgis were visualized by Galtase-YFP fluorescence and blinkout after brefeldin A addition and quantified as described (Sciaky et al., 1997). (C) Effects of two siRNAs on rate of Golgi blinkout. hsiRNA1 (expressed from pSilencer Neo) was transiently transfected into HeLa cells. Blinkout was measured as in B, 72 hours after transfection. (D) Quantification of brefeldin-A-induced blinkout in COS7 cells treated with nocodazole and brefeldin A. Cells double-transfected with CFP-Rab18-S22N and Galtase-YFP (48 hours post transfection) were placed on ice for 5 minutes in the presence of nocodozole (1 µg/ml), subsequently they were transferred to a heated microscope stage (37°C) in fresh medium containing both nocodazole and brefeldin A (5 µg/ml). Image sequences were taken as described (Sciaky et al., 1997). (E,F) Quantification of the experiments in B and C, respectively, showing median time to blinkout in cells transfected with the indicated constructs. 100-150 cells were scored for each condition.