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Fig. 6. The requirement for tGolgin-1 in microtubule minus-end-directed movement of pre-Golgi intermediates is indirect. HeLa cells transduced with Sec13-EGFP (A-F) or untransduced HeLa cells (G-J) were treated for 1 hour with 1 µg ml^–1 brefeldin A (BFA) and then washed and incubated at 16°C for an additional 3 hours in the absence of BFA. Cells were then fixed immediately or after an additional incubation at 37°C for 15 minutes to 2 hours, and analysed by IFM after labelling with antibodies to the indicated proteins and appropriate fluorochrome-conjugated secondary antibodies. Only the overlaid images from both labels are shown. (A-F) Comparison of the ER exit site marker Sec13-EGFP and either Golgi (GM130; A-C) or TGN (TGN46; D-F) elements at all three time points. (G) Comparison of Golgi (GM130) and TGN (TGN46) elements at the 15 minute time point. (H-J) Comparison of tGolgin-1 and TGN (TGN46) elements at all time points. Boxed regions in the top right corner of A,B,D,E,G-I represent 2.5 times magnified images from the corresponding panels to emphasize the degree of colocalization. Notice that, whereas GM130 initially localizes to Sec13/EGFP-containing structures before migrating towards the MTOC, TGN46 and tGolgin-1 initially localize to separate structures that remain separate during transport.

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