Fig. 5. nCEH activity as a function of native and tyrosylated HDL₃ concentrations. J774-A1 were cholesterol loaded with AcLDL (50 µg apoB/ml) as described in Materials and Methods. After an 18 hour equilibration period, the cells were incubated for 24 hours with the specified final concentrations of native (•) or tyrosylated () HDL₃. After extensive washing with PBS, cells were homogenized by sonication in 50 mM Tris-HCL buffer, pH 7.0, containing 250 mM sucrose, 1 mM EDTA, 1 mM DTT, 20 µg/ml leupeptin and 1 µg/ml pepstatin. The mixture was then ultracentrifuged for 30 minutes at 43,000 g at 4°C. The supernatant (400 µg protein, 100 µl) was assayed for nCEH activity as described in Materials and Methods. Results were expressed as a percentage of the initial activity in the absence of HDL₃. Each point represents the mean of three experiments±s.e.m. ^*P<0.05, ^**P<0.001.