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Fig. 3. Co-localisation of m-Rab_mc with markers for the prevacuolar compartment and Golgi apparatus in BY-2 cells and A. thaliana protoplasts. Antibody staining was done in BY-2 cells using FITC-labelled or Cy3-labelled secondary antibodies and in A. thaliana protoplasts expressing sialyl transferase-YFP (ST-YFP) using an Alexa568-labelled secondary antibody. Co-expression of m-Rab_mc-CFP and ST-YFP was performed in A. thaliana protoplasts. Images were colour-coded using Adobe Photoshop. Confocal images represent single images of BY-2 cells or Arabidopsis protoplasts as stated. Scale bars: 5 µm. (A-C) m-Rab_mc-Cy3/BP80-FITC dual labelling of a BY-2 cell. (A) Single image of a cell stained with m-Rab_mc antiserum. (B) Prevacuolar staining by BP80 of the same cell. (C) Merged image of the dual labelling reveals an almost complete co-localisation of the two proteins (yellow). (D-F) m-Rab_mc-Cy3/Pep12-FITC dual labelling of a BY-2 cell. (D) Single image of a cell stained with m-Rab_mc antiserum. (E) The same cell stained with Pep12. (F) Merged image shows a high level of co-localisation of the two proteins (yellow). (G-I) Single image of a BY2 cell showing m-Rab_mc-FITC/JIM84-Cy3 dual labelling. (G) m-Rab_mc-FITC; (H) Golgi-marker JIM84-Cy3; (I) merged image of the dual labelling. Note co-localisation (yellow arrowheads, inset). (J-L) Single image of an A. thaliana protoplast co-transfected with m-Rab_mc-CFP and the trans-Golgi marker ST-YFP. (J) m-Rab_mc CFP staining; (K) ST-YFP staining; (L) the merged image shows the single labelling of m-Rab_mc-stained prevacuoles (green arrowheads), ST-YFP-stained GA (red arrowheads) and co-localisation of the two proteins (yellow arrowheads). (M-O) Single image of a fixed protoplast expressing ST-YFP and labelled with m-Rab_mc antiserum. (M) m-Rab_mc staining; (N) ST-YFP staining; (O) the merged image shows the single labelling of m-Rab_mc-stained prevacuoles (green arrowheads) and ST-YFP-labelled GA (red arrowhead) and co-localisation of both proteins on some Golgi structures (yellow arrowheads).

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