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. 2010 Apr 28;285(27):20664–20674. doi: 10.1074/jbc.M109.055608

FIGURE 2.

FIGURE 2.

TMEM59 effect on APP shedding and APP maturation. A, control plasmid (con) or TMEM59 were transfected into HEK293 cells. The lysate (lys) was blotted against cellular APP (22C11, **, mature and *, immature form of the APP751 splice variant; the band below the asterisk corresponds to the immature form of the shorter APP695 variant) and TMEM59 (HA.11), the supernatant (sup) was blotted against secreted APPsα (W02). B, TMEM59 expression reduced the amount of secreted APP (APPsα) by about 60% compared with control cells. Quantification of blots shown in A. Given are mean ± S.E. of 4 independent experiments. ***, p < 0.001, determined with t test. C, control plasmid (con) or TMEM59 were transfected into HEK293 cells. The lysate (lys) was blotted against nicastrin (N1660, **, mature and *, immature form of nicastrin) and TMEM59 (HA.11). D, TMEM59 expression reduced the amount of mature nicastrin by about 30% compared with control cells. Total amounts of nicastrin were not changed. Quantification of blots shown in C. Given are mean ± S.E. of 8 independent experiments. **, p < 0.01, determined with t test. E, APP695 and either control plasmid (con) or HA-TMEM59 with GFP as transfection control were transfected into HEK293 cells. The lysate was blotted against cellular APP (22C11), TMEM59 (HA.11), and GFP, the supernatant was blotted against secreted APPsα (W02), APPsβ (192wt), and Aβ (6E10, after immunoprecipitation). F, HEK293 cells were transfected with APP695 and either control plasmid (con) or TMEM59L-HA. The experiment was carried out as described in E. G, control plasmid (con) or TMEM59 were transfected into HEK293 cells. ADAM activity was measured in intact cells for 6 h in the presence or absence of TAPI-1 (50 μm). Shown is the relative ADAM activity in % of control cells. Given are mean ± S.E. of 5 (con/TMEM59) or 3 (con TAPI-1/TMEM59 TAPI-1) independent experiments. H, BACE1 and either control plasmid (con) or TMEM59 were transfected into HEK293 cells. BACE activity was recorded for 90 min in membranes in the presence or absence of BACE inhibitor C3 (1 μm). Shown is the relative BACE activity in % of control cells. Given are mean ± S.E. of 6 (con/TMEM59) or 3 (con C3/TMEM59 C3) independent experiments.