Fig. 5.

Velocity sedimentation analysis of cytoplasmic Gag precursor complexes. 293T cells were transfected with 20 μg of the PR-inactivated versions of the indicated plasmids. Two days post-transfection, cells were homogenized and their extracted cytoplasmic lysates centrifuged through 25%, 35% and 45% sucrose step gradients at 130,000g for 1 h. Fractions were collected from gradient tops, and fraction aliquots were subjected to 10% SDS–PAGE and probed with a monoclonal antibody directed at p24CA. Total Gag proteins were quantified by scanning the immunoblot band densities of fractions 1–5. Multimerized Gag protein percentages were determined by dividing multimerized Gag protein density units (fractions 3–5) by total Gag density units (fractions 1–5) and normalizing the results to that of the wt.