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Fig. 2. Characterization of the TeM protein. (A) Detection of human TeM and ChM-I proteins expressed in COS7 cells. COS7 cells were transfected with empty vector (mock), N-terminal FLAG-tagged (FLAG-hTeM) or C-terminal FLAG-tagged (hTeM-FLAG) full-length hTeM (wild), N-terminal FLAG-tagged secreted human TeM (Glu²⁰²-Val³¹⁷) (FLAG-shTeM) and N-terminal FLAG-tagged secreted human ChM-I precursor (Glu²¹⁵-Val³³⁴) (FLAG-shChM-I). Cell lysates and culture media were analyzed by western blotting using anti-FLAG monoclonal antibodies. (B) Identification of human TeM as a transmembrane protein by cell surface biotinylation. COS7 cells transfected with empty vector (mock), FLAG-hTeM cDNA or hTeM-FLAG cDNA were biotinylated on their cell surfaces. The expressed protein was immunoprecipitated with anti-FLAG antibody and detected by streptavidin binding and anti-TeM antibody, respectively. (C,E,G) COS7 cells expressing TeM tagged with either FLAG or empty vector were incubated with FITC-conjugated anti-FLAG M2 antibody for 2 hours at 4°C under nonpermeabi lizing conditions and fixed with PBS containing 4% paraformaldehyde. (D,F,H) Permeabilized COS7 cells were incubated with FITC-conjugated anti-FLAG M2 antibody. Cells expressing hTeM-FLAG are shown in C and D, FLAG-hTeM in E and F and empty vector in G and H, respectively. Scale bars: 20 µm.

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