Inhibition of neddylation blocks Vpx mediated depletion of SAMHD1. HEK293T cells were transfected with an expression vector for FLAG–CUL4A or empty expression vector (pcDNA3.1(−)), and either treated with 250 nM MLN4924 or mock treated for four hours before harvest. Proteins were isolated from the cell lysates using beads coated with FLAG-specific antibody, and immunoblotted for either NEDD8 or the FLAG epitope as indicated (A). Monocyte derived macrophages (B) or PMA-differentiated THP1 cells (C) were treated with 1 μM MLN4924 or mock treated and infected with VSV-G-pseudotyped HIV-1, HIV-1 with a frame shift mutation in Vpr, HIV-2 or HIV-2 with a frame-shift mutation in Vpx, Vpr or both. Cells were harvested and the lysates immunoblotted for CUL4A, β-actin, SAMHD1, and HIV p24/p27. PMA differentiated THP1 cells were mock treated or treated with increasing concentrations of MLN4924 before infection with HIV-2. Cells were harvested and the lysates were analyzed by western blotting as indicated (D). HEK293T cells were transfected with either an empty expression vector (vector) or one for expression of dominant negative (DN) UBC12 and infected with VSV-G-pseudotyped HIV-2 or HIV-2 with a frame-shift mutation in Vpx. Cells were harvested and immunoblotted for CUL4A, tubulin, SAMHD1, UBC12 and HIV p24/p27 (E).