Extended Data Fig. 5: ATV:TREM2 does not promote ERK1/2 phosphorylation or a proinflammatory signature in microglia.

(a) Representative Western blot images of phosphorylation of 4EBP1 (T37/46) and ERK1/2 (T202/Y204) in WT iMGs treated for 96 h with 100 nM ATV:TREM2 or an isotype control. (b) Quantification of p4EBP1 (T37/46) and pERK1/2 (T202/Y204) normalized to actin. Relative expression was calculated by normalizing to PBS vehicle control for each experiment (n = 4 independent experiments, two-tailed paired t-test, mean ± SEM). (c) Representative Western blot images of total protein levels of mTOR and AKT in WT iMG treated for 96 h with 100 nM ATV:TREM2 or an isotype control. (d) Quantification of total mTOR and AKT protein normalized to actin. Relative expression was calculated by normalizing to PBS control for each experiment (n = 4 independent experiments, two-tailed paired t-test, mean ± SEM). e) Representative Western blot images for p-mTOR (S2448), pAKT (S473), pGSK3b (S9) pRPS6 (S235/236), p4EBP1 (T37/46) and pERK1/2 (T202/Y204) showing inhibition of mTOR pathway activation in WT iMG co-treated with 20 nM AZD8055 and 100 nM ATV:TREM2 after 96 h. (f) Quantification of phosphorylation targets shown in (E). Phosphorylation signals were normalized to actin. Relative expression was calculated by normalizing to PBS vehicle control for each experiment (n = 4 independent experiments, two-tailed paired t-test, mean ± SEM)). (g) Heatmap of human cytokine profiling in supernatant from WT iMG treated with 100 nM ATV:TREM2 for 96 h. Media collected from iMG treated with 10 ng mL⁻¹ LPS for 24 h was used for comparison.