Comparison of ability of native apoE
4 to form a denaturant-resistant complex with synthetic A
Aβ1–40 (0.5 mg/ml) was added to each of the three types of conditioned media. For media containing apoE isoforms, apoE concentration was adjusted to 20 µg apoE/ml as described in Materials and Methods. Incubations were carried out for 2 hr at room temperature. Aliquots of the reaction mixtures were separated by SDS-PAGE in a 10–20% tricine gel, electroblotted to nitrocellulose and probed with either anti-Aβ1–17 antibody 6E10 (left panel) or anti-apoE antibody (right panel). Lanes 1 and 4, conditioned medium from untransfected RAW264 cells; Lanes 2 and 5, conditioned medium from apoE ε3-transfected RAW264 cells; Lanes 3 and 6, conditioned medium from apoE ε4-transfected RAW264 cells. Arrowhead indicates the location of apoE ε3/Aβ complex which migrates as a discrete band in Lane 2 and as the slowly migrating component of the apoE ε3 band in Lane 5. Bracket indicates position of free Aβ1–40, visible in Lanes 1–3. Markings in center of figure represent positions of molecular weight standards of 200, 97.4, 69, 46, 30, 21.5, and 14.3 kD, respectively, from top to bottom.