Fig 2: 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, ß-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).

Fig 3: Neddylation is important for Vpr mediated depletion of UNG2 through CRL4. HEK293T cells were either mock treated or treated with 250 nM MLN4924 for four hours and infected with VSV-G-pseudotyped-HIV-1 or -HIV-1 with a frame shift mutation in Vpr. Twenty-four hours after infection, cells were harvested and the lysates immunoblotted for endogenous UNG2, HIV-1 p24 and tubulin (A endog.). HEK293T cells were transfected with an expression vector for UNG2 with two HA epitope tags (UNG2–2HA). Forty-eight hours later, cultures were mock treated or treated with 250 nM MLN4924 for four hours and then infected with VSV-G-pseudotyped-HIV-1 or -HIV-1 with a frame shift mutation in Vpr. Twenty-four hours later, cells were lysed and immunoblotted for the HA epitope, HIV-1 p24, and tubulin (A exog.). HEK293T cells were treated for 2 hours with concentrations of MLN4924 as indicated and then either mock infected or infected, in parallel, with a reduced MOI (approximately 2) of VSV-G-pseudotyped HIV-1. The cells were harvested for immunoblotting 24 and 48 hrs after infection. Blots were probed for endogenous UNG2, CUL4A, tubulin or HIV-1 p24 (B). Twenty-four hours after transfection with UNG2–2HA expression vector, HEK293T cells were treated and infected as described for panel B. Forty-eight hours after infection, cultures were harvested for western blot analysis and probed for UNG2–2HA (HA), CUL4A, HIV-1 p24, or tubulin (C). HEK293T cells were transfected with UNG2–2HA expression vector and either an expression vector (pcDNA3.1(-)) or ones for DN UBC12 or DN CUL4A. Twenty-four hours later cultures were mock infected or infected with VSV-G-pseudotyped-HIV-1. Twenty-four hours after infection cultures were lysed for immunoblotting and probed for UNG2–2HA (HA), endogenous UNG2, CUL4A, DN CUL4A (HA), DN UBC12, HIV-1 p24, or tubulin (D).

Fig 4: Neddylation is differentially regulated during C2C12 differentiation. (A–C) Western blot of the indicated proteins (A), and quantifications of NAE1, UBC12, and neddylated Cullins (N8-CULs) (B); quantifications of MYOD1, MYOGENIN, and MYOSIN (skeletal, fast) (MyHC) (C) in proliferative C2C12 cells cultured in growth media (i.e., day 0, D0) and for the indicated days after differentiation induction (D1 to D5). Proteins were quantified and normalized to ACTB. Data were presented as fold changes with D0 cells set at 1. N8-Conj.: NEDD8-conjugated. Arrows indicate neddylated CULs and CUL1. (D) Representative immunofluorescence images of C2C12 cells stained with NEDD8 (green) in growth media (D0) and for 3 and 6 days in differentiation media (D3, D6). Nuclei were counterstained with Hoechst 33342. Scale bars: 50 µm. ** p < 0.005 versus D0. One-way ANOVA. Three independent experiments.