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. 2014 Oct 2;6(10):3738–3765. doi: 10.3390/v6103738

Figure 3.

Figure 3

Influence of the S40N, S40D or S40A mutation on Gag ubiquitination. (A) HeLa cells were co-transfected with HA-tagged ubiquitin and pNL4-3 plasmids, as indicated. Gag processing was inhibited by the addition of the PR-inhibitor Ritonavir. The isolation and detection of ubiquitinated Gag species from cell fractions were carried out as described in Figure 2. The amount of Gag recovered by either immune precipitation or from whole cell lysates, and the VLP fraction was detected with CA specific antibodies; (B) HeLa cells were co-transfected with HA-tagged ubiquitin and the syngag expression plasmids directing the expression of wt Gag and mutants thereof. The isolation and detection of ubiquitinated Gag species from cell fractions were carried out as described in Figure 2. The amount of Gag recovered by immune precipitation, Gag recovered from whole cell lysate, and the VLP fraction was detected with CA specific antibodies.