Figure 6

Effects of the L3S and/or Y23S mutations on the chimeras ability to be retrieved from the plasma membrane. HeLa cells were cotransfected with GFP vector and with the appropriate A MLV or B MPMV chimera cDNA. Cells were then incubated for 1 hour at 4C with anti-CD25 antibody before being either shifted for 30 min at 37°C or not. Anti-CD25 stainings were revealed using phycoerythrine-conjugated secondary antibodies. Stained cells were analyzed using flow cytometry excluding the none transfected GFP-negative cells. We then plotted the percentage of internalization as the ratio between the CD25-associated fluorescence that disappeared during the 30 min uptake at 37°C and the CD25-associated fluorescence at time 0. CD25 is the reference protein without any viral cytoplasmic tail (negative control) and CD25-TFR is the CD25 reference protein in the cytoplasmic tail of which the well described YTRF endocytosis motif of the transferrin receptor has been inserted (positive control).