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Figure S1. A APPsα secretion was examined in cell culture medium collected from CHOK1 cells overexpressing APP770swe and APP770swe&Dgr;NPTY (as shown in Fig. 2, 3rd panel). B APPsβswe secretion in cell culture medium was examined (as shown in Fig. 2, 4th panel). C C99 and C83 levels were also examined in cell lysates of APP770swe and in APP770swe&Dgr;NPTY expressing cells (as shown in Fig. 2, 5th panel). D Total Aβ secretion was examined by Western blotting of cell culture medium collected from the indicated cells (as shown in Fig. 2, 6th panel). Protein bands were quantified and the data represent the mean value, calculated from at least three independent experiments, with the standard error of the mean (± SEM). APPsα, APPsβswe and Aβ data were normalised to full-length APP770swe expression levels. CTF levels were normalised to the levels of C83 expressed in APP770swe cells. Statistical analysis was carried out by using Student’s t-test. * - significance at p < 0,05, ** - significance at p < 0,01. E Aβ40 and Aβ42 were measured in 24 h conditioned media, from cells expressing different mutations within the endocytosis domain GYENPTY, with a standard ELISA. Statistical analysis was performed using one-way ANOVA and Dunett’s post tests. * - significance at p < 0,05, ** - significance at p < 0,01.

Figure S2 A APPsβwt secretion was examined in wild-type APP695myc cells, APP695&Dgr;NPTYmyc cells and APP695&Dgr;NPTYmyc cells containing an α-secretase cleavage mutation, F615P (as shown in Fig. 5A, 5th panel). B APPsβswe secretion was examined in the above mentioned cells (as shown in Fig. 5A, 4th panel). C Total Aβ levels were examined in the same cells (as shown in Fig. 5A, 7th-8th panel). Protein bands were quantified and the data represent the mean value, calculated from at least three independent experiments, with the standard error of the mean (± SEM). All data were normalised to levels of either the full-length APP695wt or the full-length APP695swe, as indicated. ** - significance at p < 0,01. E CHOKI cells stably overexpressing C99myc or C99&Dgr;NPTYmyc were treated with 10 mM NH4Cl to neutralize intracellular acidic organelles. C99, C83 and Aβ levels were examined with the indicated antibodies. F Aβ levels were quantified and normalised to the levels of C99. The data represent the mean value, calculated from at least three independent experiments, with the standard error of the mean (± SEM). ** - significance at p < 0,01.

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