Fig. 3.

Membrane flotation centrifugation of HIV-1 Gag proteins. (A) 293T cells were transfected with PR-defective versions of the indicated constructs. Two days post-transfection, cells were harvested and homogenized. Crude membrane extracted from cell lysates was subjected to equilibrium flotation centrifugation as described in Materials and methods. In all, 10 fractions were collected from top downwards; fraction aliquots were analyzed by Western immunoblotting using an anti-p24CA monoclonal antibody. During ultracentrifugation, membrane-bound Gag proteins floated to the 10–65% sucrose interface. (B) Quantification of membrane-bound Gag proteins. Total Gag proteins were quantified by scanning the immunoblot band densities of fractions 1–10. Percentages of membrane-bound Gag proteins were determined by dividing the membrane-bound Gag protein density unit (fractions 2–4) by total Gag protein density unit and multiplying by 100. Error bands indicate standard deviation. ⁎p < 0.05, ⁎⁎p < 0.01.