Figure 4.

Pulse-chase analysis of SAMHD1 in the presence or absence of Vpx. HeLa cells were transfected with 4.5 μg each of pcDNA-SAMHD1 WT, Δ14, or Δ8-9 either with 0.5 μg of empty vector DNA (no Vpx) or together with 0.5 μg of pCMV-Vpx_mac239 (Vpx(+)). Thus, the SAMHD1:Vpx DNA ratio was 10:1 for all samples. Pulse-chase analysis was performed 24 h later by labeling cells for 15 minutes with [³⁵S]-methionine/cysteine and chasing for up to 2 h as described in Methods. Labeled SAMHD1 proteins were immunoprecipitated using SAM416 antiserum, separated on SDS-PAGE, and visualized by fluorography. Protein bands were quantified by PhosphorImage analysis and plotted as a function of time. Signal intensity at the 0 time points was defined as 100%. Error bars reflect the standard error of the mean from two independent experiments.