Fig 4.

Neddylated CUL4A complexes are short lived, and high levels of Vpx cannot overcome the MLN4924-induced block to degradation. (A) MLN4924 (0.05, 0.25, 1.0, or 2.0 μM) was added to the MDDC 4 h after, at the same time as, or 2 h before the addition of VLP. After 20 h of exposure to the VLP, cell lysates were prepared and SAMHD1 was detected on an immunoblot. GAPDH was used as a loading control. The arrows denote the time of addition of the VLP, and the lightning bolts denote the time of addition of MLN4924. (B) MDDC were incubated with MLN4924, as described in the legend to panel A, for 20 h, after which the cells were lysed in urea-containing lysis buffer and CUL4A was visualized on an immunoblot probed with an anti-CUL4A antibody, with GAPDH serving as a loading control. The ratios of neddylated to unneddylated CUL4A were quantified. (C) MLN4924 was added to MDDC at the concentrations shown. After 2 h, increasing amounts of Vpx-containing or control VLP were added. After 20 h, the cells were lysed and SAMHD1 was visualized on an immunoblot probed with anti-SAMHD1 MAb and with anti-GAPDH antibody as a loading control.