Fig 1: APP processing by GAL3BP-treatments. H4-APPsw cells were treated with (+GAL3BP) or without (Cont.) the commercially available GAL3BP. a and b, sAPPα and sAPPβ proteins in the culture media and CTFs in the cell lysates were detected with Western blot analysis. Blots with β-actin were used as a loading control. The relative sAPPβ and sAPPα in the culture media from GAL3BP-treated cells were normalized to the control samples. The relative CTFs were normalized to the corresponding β-actin levels. c, the amounts of sAPPβ and sAPPα in the conditioned media and βCTF in the cell lysates were measured using ELISAs. d, the amounts of Aβ40 and Aβ42 in the lysate were measured using ELISAs. e, Western blot analysis of BACE1 and ADAM10 in the lysates of H4-APPsw cells treated with or without GAL3BP. Blots with β-actin were used as a loading control. The relative BACE1 and ADAM10 values are normalized to the corresponding β-actin levels. p > 0.05 versus control by one-way ANOVA with Tukey's post-hoc test. b–e, the controls were vehicle controls, and the values in this graph are presented relative to each control (n = 3, mean ± S.D. (error bars)). *, p < 0.05; ***, p < 0.001 versus control by one-way ANOVA with Tukey's post-hoc test.
Fig 2: Interaction of APP and GAL3BP. a–d, inhibitory effects of GAL3BP on the processing of immunoprecipitated APP by BACE1. Immunoprecipitated APP from H4-APPsw cells were treated with BACE1 (2.5 unit/ml) in the presence or absence of the commercially available GAL3BP (10 μg/ml) (a and b). Immunoprecipitated APP was also treated with BACE1 (2.5 unit/ml) and BACE1 inhibitor (50 nm) (c and d). The samples were then analyzed with Western blotting (a and c) and ELISAs to detect full-length (FL) APP and βCTF. The relative values for full-length APP and βCTF were normalized (b and d). The whole gel images are shown in Fig. S13. The values in this graph are presented relative to each other and the corresponding controls (n = 3, mean ± S.D. (error bars)). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control by one-way ANOVA with Tukey's post-hoc test. e, direct interaction of APP and GAL3BP. The immunoprecipitated APP proteins from H4-APPwt cells were incubated with the commercially available GAL3BP, and then the bound materials were analyzed with Western blotting using antibodies against APP and GAL3BP. Nonimmunoglobulin was used as a control. f, co-immunoprecipitation of GAL3BP and APP. The cDNA encoding GAL3BP with the c-Myc tag was transfected into H4-APPsw cells. The cell lysates were subjected to immunoprecipitation with anti-APP or anti-c-Myc antibodies, and then immunoprecipitated materials were analyzed with Western blotting. The blots were probed with anti-APP (top panels) and anti-GAL3BP (bottom panels) antibodies. Nonimmunoglobulin was used as a control. g and h, effect of GAL3BP on NRG1 processing. H4-APPsw cells were treated with the commercially available GAL3BP or the BACE1 inhibitor, and the cell lysates were analyzed with Western blotting using the antibody to NRG1. Blots with β-actin were used as a loading control. The values in this graph are presented relative to each other and the corresponding controls (n = 3, mean ± S.D.). *, p < 0.05 versus control by one-way ANOVA with Tukey's post-hoc test.
Fig 3: Therapeutic synergy of BE(-ST) and siBACE1 in modulating Alzheimer’s disease (AD) hallmarks in APP/PS1 mice.a Representative western blot images showing BACE1, phosphorylated tau (p-tau), and GSK3β levels in the hippocampus and cortex of BE(-ST)@siBACE1-treated APP/PS1 mice, control APP/PS1 groups, and wild-type (WT) mice. b Quantification of western blot for BACE1, p-tau, and phosphorylated GSK3β (p-GSK3β), normalized to GAPDH. The samples derive from the same experiment and that blots were processed in parallel. c Representative confocal laser scanning microscopy images showing amyloid-β (Aβ) plaque burden in the hippocampus and cortex of APP/PS1 and WT mice. Aβ plaques (green) and nuclei (Hoechst 33342, blue). Scale bars: top row, 500 µm; bottom row, 250 µm. d Nissl staining of brain sections 14 days post-treatment. Scale bar, 50 µm. ELISA results for complement activation-related pseudoallergy (CARPA) markers: e C5b9, f C3a, and g monocyte chemoattractant protein (MCP-1) in serum. Data are means ± SD (n = 3 biologically independent samples). Statistical significance was determined by two-tailed unpaired Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data in c, d are representative of two independent experiments with similar results. Source data are provided as a Source Data file.
Fig 4: BACE1 OE increases apoptosis and decreases survival after treatment. A) BACE1 OE clones E11 and G8 have increased change in caspase activity after CRT compared with CV (n=4, *p<0.05 with E11 p=0.0198, G8 p=0.0095, and KD p=0.0105). Data are presented as mean ± standard deviation. B) BACE1 OE clones have decreased change in viability by CTG assay after CRT compared with CV (n=3). Data are presented as mean±standard deviation. *p<0.05 with E11 p=0.0306 and G8 p=0.0313. C) Western blot of SNA lectin precipitation samples (whole cell lysates were used as input for comparison of loading for SNA samples). Original blots/gels are presented in Supplementary Figures S5 and S6.
Fig 5: BACE1 mRNA is significantly higher in pre-treatment biopsies of complete responders. RNAseq data was analyzed for 114 patients from pretreatment biopsies where the post-treatment response was either incomplete response (ICR) or complete response (CR). BACE1 was significantly higher in CR vs. ICR samples (p=0.02) but ST6GAL1 was not significantly different (p=0.72).
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