FIGURE 5.

Inhibition of GPIHBP1-bound LPL by ANGPTL4. RHMVECs expressing GPIHBP1 were incubated with FLAG-tagged human LPL for 3.5 h at 4 °C. After washing off unbound LPL, the indicated concentrations of ANGPTL4 were then added to the cells, and cells were incubated for an additional 10 min at 37 °C. Cells were then washed and treated with 100 units/ml heparin to release surface-bound LPL. Released LPL was then immediately assayed for lipase activity (A). Both heparin-released LPL and cell extracts were also subjected to Western blotting with the appropriate antibodies (B). C, RHMVECs expressing GPIHBP1 were likewise incubated with LPL, washed, and incubated with the indicated concentrations of ANGPTL4 for 30 min at 37 °C. Cells were then washed and treated with 5 units/ml PIPLC to release surface-bound LPL-GPIHBP1 complexes. Lipase activity was immediately assayed. For both A and C, graphs show activity (mean ± S.E.) of three independent experiments, each performed in triplicate. Means are plotted relative to control-treated LPL. Blot (B) show biological triplicates and is representative of three independent experiments.