SIV Nef inhibits Tf internalization. (A) 293 T cells expressing the indicated Nef proteins were incubated with Alexa647-conjugated Transferrin for different periods of time. Then the cells were exposed to an acidic washing procedure to remove surface bound Tf and the amount of internalized Tf was determined by flow cytometry as described in detail in the methods section. Curves and SD were generated from three independent transfection procedures. (B) THP-1 monocytes were infected with HIV-1 NIG expressing NL4-3 Nef or the SIVmac 239 Nef. 48 hours post infection cells were incubated for 5 minutes with Alexa647-conjugated Transferrin. Then surface bound Transferrin was removed by acidic washing. Subsequently cells were fixed and z-stacks of GFP and Alexa674 fluorescence were recorded by confocal microscopy. 3D reconstruction shown as maximum intensity projections (MIP) and volume calculations were done using Bitplane Imaris version 6.4. The volume ratio of internalized Tf was calculated by dividing the Tf volume through the total volume of the cell, as assessed by GFP fluorescence.