FIGURE 1.

Shedding of the TREM2 ectodomain. A, COS7 cells were co-transfected with DAP12-HA and myc-TREM2-GFP (scheme). After 24 h, cells were treated with 10 μm batimastat or dimethyl sulfoxide as solvent control for 1 h. Surface localization of TREM2 was visualized by surface staining using anti-myc primary and Alexa Fluor 594-coupled secondary antibodies (see “Experimental Procedures”). Scale bars represent 20 μm. Bar chart shows quantification of TREM2 signals in randomly chosen areas of 75 × 75 pixels (Ctrl, n = 25; batimastat, n = 40 areas). B, HEK293 cells co-transfected with FLAG-DAP12-HA and myc-TREM2-GFP were incubated in the presence or absence of batimastat (10 μm) for 24 h in serum-free medium. Cellular membranes were isolated and conditioned medium precipitated with TCA. Proteins were separated by SDS-PAGE, and TREM2 was detected by Western immunoblotting with anti-GFP (membrane) or anti-myc antibodies (medium). The different bands detected for TREM2 in membrane fractions and for the secreted TREM2 ECD in medium might represent different glycosylation state variants. Bar charts show the quantification of TREM2 ECD/FL ratios (left panel) and TREM2 CTF/FL ratios (right panel) (n = 3). Statistical analysis was done by two-tailed t test. Error bars, S.E. **, p < 0.01; ***, p < 0.001.