Fig. 8

Syt1 influences the architecture of γ-secretase complex and Aβ generation but not AICD production. a Western blot analysis of the γ-secretase complex members co-immunoprecipitated from parental and Syt1 KD PC12 cells lysed in 1 % CHAPSO (n = 4). The bottom gel shows the level of Syt1 in the input lysates and after pull-down with PS1 C-terminal antibody or IgG control. The graph presents quantitative analysis of the PS1 NTF, Pen-2 and Aph1a co-IPed with the PS1 C-terminal fragments. The intensities of the bands were measured by densitometry, and adjusted to the intensity of the input bands and to the PS1 CTF (IP efficiency control). b Quantitative analysis of the PS1 FL, PS1 NTF, PS1 CTF, and Pen-2 in total protein extracts from parental and Syt1 KD PC12 cells (n = 4 for PS1 FL, n = 8 for PS1 NTF and PS1 CTF, n = 7 for Pen-2). The intensities of the bands were normalized to β actin levels. c Cell-free γ-secretase activity assay using parental and Syt1 KD PC12 cell membrane preparations and recombinant C100-FLAG as a substrate. The amounts of Aβ40 and Aβ42 were determined by sandwich ELISA and normalized to the levels of PS1 CTF in the corresponding samples. The intensities of AICD-FLAG bands were measured by densitometry and normalized to PS1 CTF. The quantitative data in all panels are presented as mean ± SEM, n = 4. Statistical significance was determined using the unpaired student t-test, * p < 0.05, ** p < 0.01. Syt1 synaptotagmin, Aβ amyloid β, AICD APP intracellular domain, APP amyloid precursor protein, PS1 presenilin 1, NTF N-terminal fragment, Pen-2 presenilin enhancer 2, IP immunoprecipitation, CTF C-terminal fragment, FL full length