Quantitative analysis of pulse-chase experiments using transient expression in COS 1 cells.

Radiolabeled media and cell lysates were immunoprecipitated and electrophoresed on SDS-PAGE. (A) A representative experiment of wild type protein S and each mutant pulse-chase. (B) The radioactivity of protein S bands on the dried gels was measured using an image analyzer. The amount of radioactive protein S in cell lysates at the beginning of the experiment was assigned a value of 100%. The graphs represent the radioactivity recovered from cell lysates (circles), medium (squares), or total (triangles) at each time point. Total radioactivity was calculated as the sum of radioactivity recovered from media and lysate. The values represent the mean ± SD of 3 or 4 independent experiments. The P-value of an unpaired t test comparing the radioactivity in the medium at 24 hours for each mutant compared with wild type protein S is given in the right axis.