Effects of the apoE-derived peptides and the HSPG-binding peptide 6a-P on heparin binding.

<p>Heparin-immobilized beads (Pierce) were pre-equilibrated with PBS and then incubated with 500 µl of Huh-7.5 cell culture medium in the absence or presence of varying concentrations of the peptide hEP or 6a-P. The mutant peptides hEPm and 6a-Pm were used as controls. After 2 hrs incubation at room temperature, apoE-bound heparin-immobilized beads were spun down by centrifugation. The supernatant was collected and used for detection of the unbound apoE. The apoE-bound beads in pellet were washed three times with 1 ml of PBS. The heparin-bound and unbound apoE proteins were measured by Western blotting using an apoE-specific monoclonal antibody (WuE4).