or HA-beads (control, Sigma A2095, mouse) in cell lysis buffer, overnight at 4°C. For each condition, 50 µl of 50% flurry beads were first washed twice with cell lysis buffer and incubated with neuronal cell lysate collected from a whole 6-well plate of cultured human neurons (~ 4 million cells). After pull-down, the beads were washed twice with cell lysis buffer and once with kinase buffer, and then reconstituted as 50% flurry with kinase buffer. 10 µl of immunoprecipitated beads were eluted by adding 10 µl of 2X SDS-PAGE buffer. The ApoE-induced MKK7 phosphorylation and MKK7 pull-down efficiency were analyzed by immunoblotting with p-MKK7 (Cell Signaling 4171, rabbit)
