Fig. 7. Expression of FLAG-MCLT leads to clusters of SGs that colocalise with FLAG-MCLT. PC12 cells were double-transfected with hCgB-GFP(S65T) and FLAG (A-B' or FLAG-MCLT (C-E'), respectively, incubated for 2 hours at 20°C and then for 0 or 90 minutes at 37°C as indicated. Cells were fixed and immunostained against TGN38 (A-C') or the FLAG epitope (D-E'). For confocal double-fluorescence microscopy, 40 optical sections were taken for each cell. (A-C') Three-dimensional representations showing hCgB-GFP(S65T) in green and TGN38 immunostaining in red. The asterisk in B indicates the TGN of a non-transfected cell. (A-C) top views; (A'-C'), side views. Bar, 5 µm. Note that in the presence of FLAG-MCLT SGs accumulate between the TGN and the juxtaposed PM. (D-E') Single confocal section through the center of the cell (D,D') or overlay of all 40 sections (E,E'). (D,E) FLAG-MCLT-immunostaining. (D',E') Overlay of hCgB-GFP(S65T) (green) and FLAG-MCLT-immunostaining (red). Colocalising signals are shown in yellow. (F) PC12 cells were co-transfected with PTS1-GFP and FLAG-MCLT, incubated for 2 hours at 20°C and then for 90 minutes at 37°C. Thereafter cells were fixed, immunostained against the FLAG epitope and analysed by confocal microscopy. A 3D image was rendered. Green, GFP-fluorescence, red, immunofluorescence. Bar, 5 µm. See corresponding movie at http://jcs.biologists.org/supplemental.

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