Validation of the non-canonical ApoE-stimulated DLK→MKK7→ERK1/2 MAP-kinase cascade that excludes JNK activation(A) Diagram of the proposed ApoE-induced non-canonical MAP-kinase signaling pathway.(B) MKK7 inactivation by CRISPR blocks ApoE3 induction of ERK1/2 phosphorylation, while MKK7 overexpression constitutively activates ERK1/2 phosphorylation independent of ApoE3. Data are means ± SEM (n≥3 independent experiments); statistical significance (*, p<0.05, **, p<0.01; ***, p<0.001) was evaluated with one-way ANOVA and Tukey’s post-hoc test, comparing all conditions to the control without ApoE treatment. For additional data and reagent validation, see Fig. S2.(C) In vitro kinase assay with purified recombinant proteins demonstrates that ApoE3-activated MKK7 directly phosphorylates ERK2. Recombinant human ERK2 was produced in E. coli (left, stain-free SDS-polyacrylamide gel visualized by UV illumination), and naïve or ApoE-activated MKK7 was immunopurified from human neurons that overexpressed Flag-tagged MKK7 and were treated at D10 for 2 h with control or ApoE medium (center; phospho-MKK7 immunoblot). Recombinant ERK2 was then incubated for 30 min at 30 oC in the absence (test) and presence of the MEK inhibitor U0126 (50 µM; used as a further control) with Flag-beads containing immunoprecipitated control or ApoE-activated MKK7, or with control HA beads. Samples were analyzed by immunoblotting (right).
