Fig. 6. Expression of FLAG-MCLT inhibits the cortical localisation of SGs. PC12 cells were either single-transfected with hCgB-GFP(S65T) or double-transfected with hCgB-GFP(S65T) and FLAG or FLAG-MCLT, respectively, incubated for 2 hours at 20°C and then at 37°C as indicated. Cells were fixed, stained with phalloidin-TRITC for F-actin and then analysed by confocal double-fluorescence microscopy. For each cell, 40 optical sections were taken. (A,B) Overlays of single optical sections for hCgB-GFP(S65T) (green) and F-actin (red) from cells double-transfected with hCgB-GFP(S65T) and FLAG (A) or FLAG-MCLT (B), respectively, and fixed after 1 hour of chase. SGs colocalising with F-actin are indicated by arrowheads, non-colocalising SGs are indicated by arrows. Asterisks, nontransfected cells. Bar, 5 µm. (C) Quantitation of colocalisation. The 40 optical sections per colour channel were rendered with IPLab 3D-software to 3D representations (see Materials and Methods) and the percentage of SGs colocalising with F-actin was determined. The graph shows mean values for at least four cells per time point and condition from two independent experiments. n, number of SGs evaluated per condition. Error bars, s.e.m.

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