For in vitro kinase assays determining whether ApoE-activated MKK7 phosphorylates ERK2, 200 ng of recombinant human ERK2 was added into immunoprecipitated beads pelleted from 40 µl 50% flurry: Flag-beads pulling down unactivated or ApoE-activated Flag-MKK7, or control HA-beads. The reactions were done in 40 µl kinase buffer supplemented with 0.1 mM ATP at 30°C for 30 minutes, without or with U0126 50 µM. The reaction were terminated by pelleting down beads and adding 10 µl 5X SDS-PAGE buffer into the harvested supernatants (~40 µl). The phosphorylation of ERK2 was analyzed by running 20 µl of each reaction mixture supernatants for SDS-PAGE and immunoblotting using anti-ERK (Cell Signaling 4695) and anti-p-ERK (Cell Signaling 9106) antibodies.
