Fig 2.

MLN4924 prevents the Vpx-induced degradation of SAMHD1. (A) MLN4924 (0.05, 0.25, 1.0, or 2.0 μM) was added to MDDC from two donors. After 2 h, Vpx-containing (VLP X+) or control VLP (VLP X−) were added. The next day, cell lysates were prepared and SAMHD1 was visualized on an immunoblot probed with anti-SAMHD1 MAb and anti-GAPDH antibody as a loading control. (B) Differentiated U937-HA.SAMHD1 (left) and THP1 (right) cells were pretreated with MLN4924, as described in the legend to panel A, for 2 h, and Vpx-containing or control VLP were added. The next day, lysates were prepared and the SAMHD1 was visualized on an immunoblot probed with an anti-HA MAb (U937.HA-SAMHD1) or an anti-SAMHD1 MAb (THP1) and with anti-GAPDH antibody as a loading control. SAMHD1 levels were quantified relative to the GAPDH level, and the ratio of the DMSO control treated with control VLP was set to 1. (C) HeLa cells stably expressing FLAG-/HA-tagged SAMHD1 were incubated with DMSO or MLN4924 for 16 h or 40 h. Untreated cells and cells treated for 16 h with MG132 served as controls. Lysates were prepared, and SAMHD1 was visualized on an immunoblot probed with anti-HA MAb and anti-GAPDH MAb as a loading control.