FIGURE 5.

DAP12 stabilizes TREM2-CTF by suppressing its degradation. A, HEK293T cells stably expressing HA-TREM2-Myc (scheme) were transfected with wild-type (DAP12 WT-GFP) or mutant (DAP12 D50A-GFP) DAP12 plasmids. Conditioned medium was collected 24 h post-transfection and precipitated with trichloroacetic acid (TCA). Cells were harvested and the lysates were analyzed by Western blotting. sTREM2 in the conditioned medium was detected by Western blotting with an anti-HA antibody. The levels of TREM2 and DPAP12 in the cell lysates were detected by antibody against the Myc or GFP epitope, respectively. Bar graph to the right shows the relative protein level of sTREM2 in the medium (n = 3). ns, not significant. B, HEK293T cells were transfected with TREM2-Myc in combination with vector control or DAP12-GFP. After 16 h, cells were incubated with 500 nm Compound E or vehicle control. Bar graph to the right shows the quantification of Western blots as ratios of TREM2-CTF/TREM2-FL, which was calculated by subtracting the TREM2-CTF/TREM2-FL ratio in the absence of Compound E from the TREM2-CTF/TREM2-FL ratio in the presence of Compound E (n = 3). ns, not significant. C, HEK293T cells were co-transfected with TREM2-Myc and wild-type (DAP12 WT-GFP) or mutant (DAP12 D50A-GFP) DAP12 plasmids. Cells were treated with 500 nm CHX for indicated time periods (hour, hr). Bar graphs to the right show the relative levels of TREM2-FL or TREM2-CTF (n = 3). The relative signal intensities of TREM2-FL and TREM2-CTF at various time points were normalized to the “0” time point. ***, p < 0.001.