Fig. 4. GBF1 accumulates on membranes of the Golgi complex and peripheral VTCs upon treatment with BFA. (A) Images of NRK cells expressing GFP-GBF1 were captured every 2 seconds for 10 minutes immediately after BFA addition (1 µg ml^-1). Still images from indicated time points illustrate the transient accumulation of GBF1 onto peripheral VTCs and Golgi complex. (supplementary material Movie 1). Bar, 5 µm. (B) NRK cells treated with BFA (5 µg ml^-1) for 30 seconds were fixed and processed for standard IF using Alexa488-conjugated anti-GBF1 rabbit antibody (H-154) and rabbit anti-p58 antibodies. Merged image shows threefold magnification of boxed area in red and green channels. Bar, 5 µm. (C) NRK cells treated with 5 µg ml^-1 BFA for 1 minute were fixed and processed for confocal IF using polyclonal antibodies against GBF1 (H154) and BIG1 (Alexa488-conjugated 9D3). Bar, 10 µm. (D) Quantification of experiment 4A showing Golgi/total ratio plotted against treatment length. (E) NRK cells were washed in ice-cold buffer containing either DMSO vehicle control (0), or 0.5 µg ml^-1 (0.5) or 5 µg ml^-1 (5.0) BFA. Cytosol (C) and microsomes (M) fractions were prepared and analyzed for GBF1 content by immunoblot (see Materials and Methods).