Flag-tagged human MKK7 proteins were expressed in pure human neurons (iN cells) by lentiviral transduction (day 4 to day 10), and activated by ApoE stimulation (10 µg/ml for 2 hours on day 10) as described earlier. The gene fragment of human MKK7 (NM_001297555.1) was synthesized and cloned into AgeI and EcoRI sites on FUGW vector (Addgene plasmid # 14883). After mock or ApoE stimulation, human neurons were lysed with non-denaturing cell lysis buffer and brief sonication. Cell lysis buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM PMSF added immediately before use. The immunoprecipitation of unactivated or ApoE-activated Flag-MKK7 was performed by incubating cell lysates of mock- or ApoE-stimulated human neurons with Flag-beads (Sigma A2220, mouse) 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
