Figure 1.

Unsaturated 14-carbon fatty acids alter the behavior of Gag on Optiprep density gradients. (A) After overnight treatment with fatty acid-supplemented medium, Dounce homogenates of COS-1 cells transfected with pCMV5 Gag were floated in a discontinuous Optiprep gradient. Fractions were collected such that fraction 1 is the top and fraction 8 is the bottom of the gradient. Trichloroacetic acid precipitates of each fraction were subjected to SDS/PAGE and Western blotting by using anti-p24CA antibody. The percent of total Gag protein in each fraction was quantitated from cells treated with 14:0 (closed squares), 14:1n-9 (open squares), or 14:2n-6 (open diamonds). (B) Cells expressing GagΔp15 were treated with fatty acid-supplemented medium and lysed at 4°C in 0.5% TX-100. The lysates were floated on discontinuous Optiprep gradients. Five fractions were collected from the top; fraction 1 contains DRMs and fraction 5 contains the soluble lysate (SL). Closed bars represent the percent of total GagΔp15 detected in each fraction from cells treated with 14:0, hatched bars 14:1n-9, and open bars 14:2n-6. (C) GagΔp15-expressing cells were treated with 14:0 or 14:2n-6 overnight and labeled the following day with IC13. After labeling, the cells were lysed and floated as described for B. Anti-p24CA immunoprecipitates were analyzed by SDS/PAGE, and Gag-specific bands were visualized by PhosphorImager analysis. Representative gradients are depicted, showing that approximately equal fractions of IC13-labeled GagΔp15 are recovered in DRMs in either condition. (D) A comparison of radiolabel (IC13) and Western blot (WB) signals from immunoprecipitates of the same gradient of 14:2n-6-treated cells. Only the DRM and soluble lysate fractions are shown.