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Fig. 6. Internalisation of surface-labelled L6-Ag and CD63. (A) MCF-7 cells were transiently transfected with the plasmid encoding HA–L6-Ag. 48 hours later, cells were surface labelled with the anti-L6-Ag mAb (L6, IgG2a) for 1 hour at 4°C and then placed to 37°C for the indicated durations. Cells were subsequently fixed and permeabilised as described in the legend to Fig. 2. The internalised mAbs were visualised with Alexa-Fluor-594-conjugated goat anti-mouse IgG2a (red). Late endocytic organelles were visualised with anti-Lamp2 mAb and Alexa-Fluor-488-conjugated goat anti-mouse IgG1 Ab (green). (B) MCF-7 cells were prepared for the experiments as described in A except that they were incubated with the anti-CD63 mAb (6H1, IgG1) instead of the anti-L6-Ag mAb. The internalised mAbs were visualised with Alexa-Fluor-488-conjugated goat anti-mouse IgG1 Ab (green). Late endocytic organelles were visualised with the anti-HA mAb (F7, IgG2a) and Alexa-Fluor-594-conjugated goat anti-mouse IgG2a Ab (red). Scale bars: 10 µm.

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