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Fig. 1 | Molecular Neurodegeneration

Fig. 1

From: Tetraspanin 6: a pivotal protein of the multiple vesicular body determining exosome release and lysosomal degradation of amyloid precursor protein fragments

Fig. 1

TSPAN6 affects the normal proteostasis of the cell. a TSPAN6 was transfected in HEK-APPwt (n = 3 technical repeats) and analyzed by western blot using antibodies against APP, APLP2 and LRP1. The CTF levels of APP (APP-CTF), APLP2 (APLP2-CTF) and LRP1 (LRP1-CTF) were elevated by overexpressing TSPAN6. No changes in the levels of full-length proteins for APP (APP-FL), APLP2 (APLP2-FL) and LRP1 (LRP1-FL) were observed. b Quantification of bands in panel a confirmed the increase in CTF levels of APP, APLP2 and LRP1. c Aβ determination by ELISA in the 24 h conditioned medium of HEK-APPwt cells like those in panel a shows increased Aβ40 levels (n = 3 experiments). d Cell lysates of HEK-APPwt cells treated 48 h with a scrambled siRNA (siRNA-cnt) or a siRNA against TSPAN6 (siRNA-TSPAN6) were analyzed by western blot as in panel a (n = 3 experiments). In contrast to panel a, the levels of APP-CTF, APLP2-CTF and LRP1-CTF were strongly reduced in siRNA-TSPAN6-treated cells. e Quantification of bands in panel d. f Aβ levels determined by ELISA in the 24 h-conditioned medium of the cells in panel d show a significant decrease in Aβ40 levels. g HEK293 cells were co-transfected with FlagAPP-C99 and TSPAN6 or an empty vector and lysates analyzed by western blot with antibodies against FlagM2 and 6E10 (n = 3 experiments). h Quantification of FlagAPP-C99 from panel g. i Aβ in the 24 h conditioned medium of the cells from panel g measured by ELISA. TSPAN6 overexpression increases Aβ40 and Aβ42 levels. j Transfected HEK293 analyzed by pulse-chase labeling. Cells were incubated for two hours with 35S-Met/Cys and FlagAPP-C99 was immunoprecipitated at specific time-points with FlagM2-affinity gel. The density of the bands in the autoradiography was quantified and plotted (n = 3 experiments). This shows a slower disappearance of FlagAPP-C99 during the first 30 min in cells overexpressing TSPAN6. k HEK293 cells co-transfected with myc-tagged NotchΔE and TSPAN6 or an empty vector (n = 3 experiments). TSPAN6 overexpression do not change myc-NΔE levels. l Quantification of panel k shows no differences in terms of NICD production in TSPAN6. Statistical significance was determined by t-test (*p <0.05, **p <0.01, ***p <0.001)

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