Extended Data Fig. 4: EBP1 proteolysis occurs in AD brains.
From: EBP1 potentiates amyloid β pathology by regulating γ-secretase
a, Serial-aged WT mouse brains were isolated and used for the comparison of EBP1 expression pattern between RNA and protein via PCR and immunoblots, respectively. n = 4 for IB and n = 6 for PCR. b, Brain lysates of human postmortem AD patients (n = 4) and age-matched controls (n = 4) were subjected to immunoblots with anti-EBP1 and anti-HSP70 antibodies. c, Brains of 8-month WT (n = 3) and 5X-FAD (n = 3) mice were applied to a cleavage assay in pH 7.4 cleavage buffer for 45 min. Two cleaved EBP1 fragments were only detected in 5X-FAD brain lysates with anti-EBP1 antibody. d, Hippocampal neurons at DIV7 applied Aβ peptide at 1 and 10 μM, and control peptide for 24 h were stained with indicated antibodies. White boxes on the left side indicate the soma of stained neurons. Scale bar, 20 μm and 5 μm, each. e, Brains of 8-month WT and 5X-FAD mice were isolated and subjected to IHC against anti-EBP1 (red), anti-EEA1 (blue), and anti-LAMP1 (green) antibodies. Analysis of the intensity of EBP1 with EEA1 and LAMP1 signals (right). Scale bar, 20 μm. f, Brains of 3 WT and Lgmn(-/-) mice were subjected to cleavage assay and cleaved EBP1 was detected by anti-EBP1 antibody. n = 3. g, Series of EBP1 N Mutants cleavage was quantified to the bar graphs. n = 4. Data are presented as mean values ± SEM (a, f, g). *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001 (f, g), ns; not significant. Student’s two-tailed unpaired t-test (f) and Two-way ANOVA followed by Tukey’s multiple comparison (a, g).
