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Fig. 9. The role of L6-Ag in the surface expression of tetraspanins and integrins. (A) BT549 cells were electroporated with control siRNA (si-Cont) or siRNA that targets L6-Ag (si-L6Ag). The surface expression of proteins was analysed by flow cytometry after 72 hours. Data are presented as ratios of means of fluorescence intensity (MFIs) for cells transfected with control siRNA to those transfected with siRNA that targets L6-Ag. Bars represent the mean ± s.d. from three independent experiments. (B) MCF-7 cells were transfected with the plasmids encoding GFP-L6-Ag, GFP-L6K^5,86 or GFP alone. 24 hours later cells were detached and re-plated in the EMEM/10% FCS for a further 24 hours. The surface expression of proteins was analysed by flow cytometry. Data presented as ratios of means of fluorescence intensity (MFIs) for cells expressing GFP-proteins to those of non-transfected cells. Bar values represent the mean ± s.d. from three independent experiments. (C) Lysates prepared from cells transfected with control siRNA and siRNA that targets L6Ag (as described in A) were resolved in 11% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with anti-CD151 polyclonal Ab, the anti-L6-Ag mAb (L6pke), anti-CD82 mAb (TS82) and anti-CD63 mAb (1C5). Shown are the results of a representative experiment. (D) BT549 cells were electroporated with control siRNA or siRNA that targets L6-Ag. Intracellular distribution of proteins after 72 hours was analysed as described in Fig. 2. Note that CD63-positive vesicles are more evenly scattered through the cytoplasm in cells in which the expression of L6-Ag was knocked down by siRNA than controls. (E) BT549 cells were electroporated with control siRNA or siRNA that targets L6-Ag. 72 hours later cells were surface labelled with the anti-CD63 mAb 6H1 for 1 hour at 4°C and then placed to 37°C for the indicated durations. Non-internalised mAbs were labelled with IRDye-800CW-conjugated goat anti-mouse IgG. Fluorescent signals were detected using Odyssey infrared imaging system. Data is presented as percentage of the mAb 6H1 left on the cell surface relative to that at time point 0, t=0 (100%, fluorescent signals before cell were placed to 37°C) and are shown as mean ± s.d. calculated from at least three separate experiments each performed in triplicate. RFU t=N, relative fluorescence units at a given time interval; RFU t=0, relative fluorescence units at time point 0. Scale bar: 10 µm.

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