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Figure 5


Fig. 5. Removal of the GPI anchor impedes CD317 internalisation. (A) H4IIE cells were incubated for 1 hour at 37°C in serum-free medium with or without PI-PLC as indicated, and subjected to anti-CD317 antibody uptake for 20 minutes at 37°C prior to fixation and processing for immunofluorescence analysis. Bars, 10 µm. (B) Immunoblot analysis of fractions from sucrose-density-gradient separation of H4IIE cell lysates from control cells (Control) and from cells that had been incubated in PI-PLC prior to lysis (PIPLC). Fractions were taken from the top of the gradient (i.e. fraction 1 is the most buoyant), and blots were probed with CD317 antibody. (C) Surface proteins of HeLa cells were treated with PI-PLC or serum-free medium and then biotinylated. Endocytosis was allowed to proceed for 0, 2, 4 or 8 minutes after which surface biotin was removed. Internalised proteins were pulled down with streptavidin beads and analysed by western blotting with antibodies against transferrin receptor and CD317.





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