Journal: British Journal of Pharmacology
Article Title: Novel multifunctional iron chelators of the aroyl nicotinoyl hydrazone class that markedly enhance cellular NAD + /NADH ratios
doi: 10.1111/bph.14963
Figure Lengend Snippet: The effect of SNH2 and SNH6 on the protein levels of APP, BACE1, CTFα, CTFβ, TfR1, and sAPPβ in human primary astrocyte cultures, as determined by western blotting. (a) Diagram of the non‐amyloidogenic and amyloidogenic processing of APP. The non‐amyloidogenic processing of APP is mediated by α‐secretase, which cleaves APP within the Aβ domain and leads to soluble APPα (sAPPα) and the C‐terminal fragment α (CTFα). Subsequent cleavage of CTFα by γ‐secretase leads to the formation of the p3 peptide and the APP intracellular domain (AICD). The amyloidogenic processing of APP, mediated by β‐secretase, leads to the generation of soluble APPβ (sAPPβ) and the C‐terminal fragment β (CTFβ). Subsequent cleavage of CTFβ by γ‐secretase leads to the release of Aβ and AICD formation. (b) Human primary astrocyte cells were incubated with media containing batimastat (5 μM), C3 (5 μM), SNH2, or SNH6 (25 μM) for 24 hr at 37°C. Cell lysates and media were collected and used for western blot analysis. Densitometry of (c) APP; (d) BACE1; (e) CTFβ; (f) CTFα; (g) TfR1 (control for cellular Fe depletion); and (h) sAPPβ. Results shown are mean ± SD (N = 5 biological replicates). *P < .05, significantly different from control. (i) The effect of SNH2 and SNH6 on Aβ1–40 levels. Aβ1–40 release in the culture medium after a 24 hr/37°C incubation of human primary astrocytes with batimastat (5 μM), C3 (5 μM), SNH2 (25 μM), or SNH6 (25 μM) was quantified using an ELISA sandwich kit. Results shown are mean ± SD (N = 6 biological replicates). *P < .05, significantly different from control [Colour figure can be viewed at http://wileyonlinelibrary.com]
Article Snippet: The following primary antibodies were used in this study: β‐site APP cleaving enzyme 1 (BACE1) rabbit mAb (catalogue #5606T; rabbit IgG; Epitope: residues surrounding H490 of human BACE1) and APP/Aβ (NAB228) mouse mAb (catalogue #2450; mouse IgG2A; Epitope: N ‐terminus of Aβ) from Cell Signaling Technology; anti‐APP C ‐terminal rabbit pAb (catalogue #A8717; rabbit IgG; Epitope: C ‐terminal of human APP) and anti‐β‐actin mouse mAb (catalogue #A5316; mouse IgG2a; Epitope: N ‐terminal of β‐actin; RRID:AB_476743) from Sigma‐Aldrich; purified anti‐sAPPβ rabbit pAb (catalogue #813401; rabbit IgG; Epitope: N ‐terminus of sAPPβ) from Biolegend and transferrin receptor 1 (TfR1) mouse mAb (catalogue #13‐6890; mouse IgG1; Epitope: N ‐terminal region of TfR1) from Thermo Fisher Scientific.
Techniques: Western Blot, Incubation, Control, Enzyme-linked Immunosorbent Assay
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Thermo Fisher manufactures this product 
![The effect of SNH2 and SNH6 on the protein levels of APP, BACE1, CTFα, CTFβ, <t>TfR1,</t> and sAPPβ in human primary astrocyte cultures, as determined by western blotting. (a) Diagram of the non‐amyloidogenic and amyloidogenic processing of APP. The non‐amyloidogenic processing of APP is mediated by α‐secretase, which cleaves APP within the Aβ domain and leads to soluble APPα (sAPPα) and the C‐terminal fragment α (CTFα). Subsequent cleavage of CTFα by γ‐secretase leads to the formation of the p3 peptide and the APP intracellular domain (AICD). The amyloidogenic processing of APP, mediated by β‐secretase, leads to the generation of soluble APPβ (sAPPβ) and the C‐terminal fragment β (CTFβ). Subsequent cleavage of CTFβ by γ‐secretase leads to the release of Aβ and AICD formation. (b) Human primary astrocyte cells were incubated with media containing batimastat (5 μM), C3 (5 μM), SNH2, or SNH6 (25 μM) for 24 hr at 37°C. Cell lysates and media were collected and used for western blot analysis. Densitometry of (c) APP; (d) BACE1; (e) CTFβ; (f) CTFα; (g) TfR1 (control for cellular Fe depletion); and (h) sAPPβ. Results shown are mean ± SD (N = 5 biological replicates). *P < .05, significantly different from control. (i) The effect of SNH2 and SNH6 on Aβ1–40 levels. Aβ1–40 release in the culture medium after a 24 hr/37°C incubation of human primary astrocytes with batimastat (5 μM), C3 (5 μM), SNH2 (25 μM), or SNH6 (25 μM) was quantified using an ELISA sandwich kit. Results shown are mean ± SD (N = 6 biological replicates). *P < .05, significantly different from control [Colour figure can be viewed at http://wileyonlinelibrary.com]](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1547/pmc07161547/pmc07161547__BPH-177-1967-g006.jpg)