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Fig. 1 | Molecular Neurodegeneration

Fig. 1

From: Amyloid-beta modulates microglial responses by binding to the triggering receptor expressed on myeloid cells 2 (TREM2)

Fig. 1

Oligomeric Aβ1–42 specifically binds to TREM2 and activates TREM2 signaling pathway. a Schematic representation of human TREM2 extracellular domain (sTREM2, amino acid residues 1–171) tagged with human IgG1 Fc. SP: signal peptide. b The cDNA encoding sTREM2-Fc, sTREM1-Fc or Fc alone was transfected into HEK 293 T cells. Each protein was purified from the conditioned medium and analyzed by silver stained SDS-PAGE. c The prepared oAβ1–42 peptides were analyzed by Western blotting using 4–12% Bis-Tris NuPAGE gel. d Solid phase binding assay showing the saturation binding curve and equilibrium dissociation constant (KD) of oAβ1–42 binding to sTREM2-Fc. Fc and sTREM1-Fc served as negative controls (n = 3). e The Fc, sTREM2-Fc or sTREM1-Fc control was pre-bound to protein A agarose beads and used as baits for immunoprecipitation of oAβ1–42. The precipitated products were separated on 4–12% Bis-Tris NuPAGE gel and further subjected to Western blotting. (f and g) The binding profiles of oAβ1–42 to different concentrations of sTREM2-Fc f or Fc g were generated by SPR assay. h The prepared monomeric Aβ1–42 (mAβ1–42) peptides were analyzed by Western blotting using 4–12% Bis-Tris NuPAGE gel. i The binding profiles of mAβ1–42 to different concentrations of sTREM2-Fc were generated by SPR assay. jThe binding profiles of scrambled Aβ42 (scAβ42) to different concentrations of sTREM2-Fc were generated by SPR assay. k Wild-type (WT) or Trem2-knockout (KO) microglia were stimulated with 1.0 μM oAβ1–42 for 1 h. Cell lysates were analyzed by Western blotting using antibodies specific for either total (T-Syk) or phosphorylated form (p-Syk) of Syk. l Quantification of Western blots as ratios of p-Syk/T-Syk. β-Actin was used as an internal control (n = 3, two-way ANOVA). m WT or Trem2-KO microglia were stimulated with 1.0 μM oAβ1–42 for the indicated time. Cell lysates at each time point were analyzed by Western blotting using antibodies specific for either total (T-Akt) or phosphorylated form (p-Akt) of Akt. n Quantification of Western blots as ratios of p-Akt/T-Akt. β-Actin was used as an internal control (n = 3, two-way ANOVA). Data information: Data represent mean ± SD. **, p < 0.01; ***, p < 0.001; ns, not significant

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