FIGURE 3.

Half-life of endocytosed NEU1 in Ppca^−/− and Ppca^+/+ macrophages. Macrophages were maintained in culture in 6-well plates in the presence of either purified NEU1 alone (25 μg) or a mixture of BV-NEU1 and BV-PPCA (25 μg each) for 16 h. The medium was then replaced with enzyme-free medium. Cells were harvested at 0, 1, 3, 5, 7, and 24 h and assayed for NEU1 activity (A). NEU1 uptake was measured in Ppca^+/+ macrophages (red plot) or Ppca^−/−macrophages (blue plot), and PPCA-NEU1 uptake was measured in Ppca^−/− macrophages (black plot). NEU1 activity is expressed as nmol of substrate/h/mg of total protein. B–E, harvested cell lysates were also analyzed on immunoblots with anti-PPCA and anti-NEU1 antibodies.