ApoE was removed from HDL by heparin-sepharose chromatography, then the cholesterol efflux studies were using the holo–HDL-2 fraction or HDL-2 depleted of apoE. (A) Immunoblot analysis of LCAT, apoE, and apoA-I in the holo–HDL-2 fraction and the apoE-free HDL-2. (B) Isotopic CE formation in media containing holo–HDL-2 or apoE-free HDL-2. THP-1 macrophages were incubated in RPMI-1640 containing [³H]AcLDL with T0 for 24 hours, then cholesterol efflux was performed for 8 hours in RPMI-1640 containing HDL-2 (50 μg/ml HDL protein). The data represent mean ± SD of an experiment performed in triplicate. ^†P < 0.001, versus apoE⁺ control HDL-2. (C) Net cholesterol efflux to holo–HDL-2- and apoE-free HDL-2 from THP-1 macrophages. Macrophages were treated with T0 and AcLDL for 24 hours, and then cholesterol efflux was performed for 8 hours in RPMI-1640 containing HDL-2 (50 μg/ml HDL protein). The data represent mean ± SD of the experiment performed in triplicate. *P < 0.05, **P < 0.01, versus apoE⁺-control HDL-2. (D) LCAT activity of HDL-2 particles. FC content of HDL-2 fraction was measured using the enzymatic method before and after incubation in plastic tubes for 8 hours at 37°C with gentle shaking. The reduced FC mass of HDL-2 was estimated as the actual LCAT activity. The data represent mean ± SD of an experiment performed in triplicate. *P < 0.05, ^#P < 0.005 versus 0 hours.