Extended Data Fig. 5: AEP-mediated EBP1 cleavage is determined by either anti-N84 or N204 antibody.
From: EBP1 potentiates amyloid β pathology by regulating γ-secretase
a, A diagram illustration of N84- and N204-specific antibody production process. b-c, Antibody specificity test. b, In vitro cleavage assay with purified GST-EBP1 and rAEP proteins. Both the anti-N84 and anti-N204 antibodies detected the correct size of cleaved EBP1 with an anti-GST antibody. c, GST-tagged EBP1 fragments were transfected to HEK293T cells. Cells were lysed and conducted to IB with anti-GST antibody, anti-N84 and N204 antibodies. Red arrow: cleaved EBP1. d, Brains of 5X-FAD mice at serial ages by IHC with anti-Aβ (green) and anti-N84 or N204 antibodies (red). Nuclei were stained with DAPI. Scale bar, 100 μm. e, An indicated series of 5X-FAD brain lysates were subjected to IB for detecting the N84 and N204 fragments as aging. The intensity of N84 and N204 cleavage forms was quantified (right). n = 8. f, Hippocampus and cortex of 8-month 5X-FAD and WT mice immunostained with anti-EBP1 or N84 or N204 antibody (red) with anti-NeuN (green) antibody. Nuclei were stained with DAPI. Quantification of fluorescence (bottom). Scale bar, 50 μm. n = 5. g, Brains of 10-month control mice stained with anti-N84 or N204 antibodies. h, Peptide inhibition assay for exploring antibody specificity. 5-month 5X-FAD tissues were incubated with N84 or N204 peptides or control peptide as control. After incubation, tissue samples were stained with indicated antibodies. Scale bar, 100 μm. Data are presented as mean values ± SEM (e, f). *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001 (e, f), ns; not significant. Multiple unpaired t-test (f) Two-way ANOVA followed by Tukey’s multiple comparison (e).
