Overexpression of ALIX increases virus release but has no influence on Gag ubiquitination. ( A ) HeLa cells were co-transfected with HA-tagged ubiquitin, pΔR constructs as indicated, and either with, or without, FLAG-ALIX plasmid. Gag processing was inhibited by the addition of the PR-inhibitor Ritonavir. For detection of ubiquitinated Gag species, Gag was precipitated from whole cell lysates by immunoprecipitation using anti- HIV antibodies, and ubiquitin was detected by anti- HA staining. VLPs were pelleted from the supernatant and the amount of Gag released as VLPs was detected by anti- CA staining; ( B ) Evaluation of three independently performed experiments. Bars represent the arithmetic mean ± SD. The numbering is according to (A).