Journal: bioRxiv

Article Title: Alzheimer-mutant γ-secretase complexes stall amyloid β-peptide production

doi: 10.1101/2024.08.30.610520

Figure Lengend Snippet: (A) Design of fluorescence lifetime imaging microscopy (FLIM) set-up to detect E-S complexes of γ-secretase and C99/Aβ-intermediates. 6E10-Alexa Fluor™ 488 over C99-720 fluorescence ratio (6E10-A488/C99-720 ratio) enables distinguishing C99-rich and Aβ-rich subcellular compartments. (B) PSEN1/2 dKO HEK293 cells were co-transfected with C99-720 and WT or FAD mutant PSEN1. Transfected cells were immunostained with anti-C99/Aβ (mouse 6E10) and anti-nicastrin (rabbit NBP2-57365) primary antibodies and Alexa Fluor™ 488 (FRET donor) or Cy3 (acceptor)-conjugated anti-mouse and anti-rabbit IgG secondary antibodies, respectively. The donor 6E10-Alexa Fluor™ 488 (6E10-A488) lifetime was measured by FLIM. Energy transfer from the donor to the acceptor results in shortening of the donor lifetime. Scale bars, 10 µm. (C) 6E10-A488 lifetimes were analyzed in randomly selected ROIs (N=40-47 from 6-8 cells), highlighting increased E-S complexes in the cells with FAD PSEN1 mutants, except F386S, compared to WT controls. One-way ANOVA and Tukey’s multiple comparisons test; n.s., p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (D) Representative images of confocal, pseudo-color analysis to identify C99 or Aβ-rich subcellular areas and corresponding FLIM in wild-type or F386S PSEN1 expressing cells. Scale bars, 10 µm. (E) In the areas with lower 6E10-A488/C99-720 ratios (i.e., C99-rich areas), 6E10-A488 lifetimes were not different between the cells with WT PSEN1 and those with F386S mutant. N=21 ROIs. (F) On the other hand, 6E10-A488 lifetimes were significantly shorter in the cells expressing F386S mutant PSEN1 compared to wild-type controls in Aβ-rich ROIs (N=23). Unpaired T-test **p < 0.01.

Article Snippet: The following antibodies were used: Anti-Nicastrin (Novus Biologicals, NBP2-57365), Anti-PSEN-1-NTF (Bio-Legend, 823401), anti-PSEN-1-CTF (Cell Signaling, 5643), Anti-Aph-1 (Bio-Legend, 823101), and Anti-Flag M2 (Sigma-Aldrich, F1804).

Techniques: Fluorescence, Imaging, Microscopy, Transfection, Mutagenesis, Expressing

Journal: eLife

Article Title: Alzheimer-mutant γ-secretase complexes stall amyloid β-peptide production

doi: 10.7554/eLife.102274

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit anti-nicastrin , Novus Biologicals , Cat. No. NBP2-57365 , .

Techniques: Expressing, Transfection, Western Blot, Stripping, Recombinant, Synthesized, Mutagenesis, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Gel Extraction, Sequencing, FLAG-tag, Software

Journal: eLife

Article Title: Alzheimer-mutant γ-secretase complexes stall amyloid β-peptide production

doi: 10.7554/eLife.102274

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: Anti-Nicastrin (Novus Biologicals, NBP2-57365), Anti-PSEN-1-NTF (Bio-Legend, 823401), anti-PSEN-1-CTF (Cell Signaling, 5643), Anti-Aph-1 (Bio-Legend, 823101), and Anti-Flag M2 (Sigma-Aldrich, F1804).

Techniques: Expressing, Transfection, Western Blot, Stripping, Recombinant, Synthesized, Mutagenesis, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Gel Extraction, Sequencing, FLAG-tag, Software