Journal: Journal of Neuroinflammation

Article Title: Involvement of 5-lipoxygenase activating protein in the amyloidotic phenotype of an Alzheimer’s disease mouse model

doi: 10.1186/1742-2094-9-127

Figure Lengend Snippet: In vitro effect of MK-591 on Aβ formation and amyloid-β precursor protein metabolism. N2A-APPswe cells were incubated with increasing concentration of MK-591 or vehicle for 24 h, and conditioned media and cell lysates collected. (A) Aβ1-40 levels in the supernatant assayed by sandwich ELISA (n = 4 per each condition; * P <0.01). (B) Representative western blots of APP, ADAM-10, BACE-1, PS1, nicastrin, APH-1, and Pen-2 in the lysates of MK-591 or vehicle-treated cells. (C) Densitometric analyses of the immunoreactivities to the antibodies shown in panel B (* P <0.01). ADAM-10: disintegrin and metalloproteinase domain-containing protein 10; APH-1: anterior pharynx-defective 1; APP: amyloid-β precursor protein; BACE-1: β-site amyloid precursor protein cleaving enzyme 1; ELISA: enzyme-linked immunosorbent assay; N2A-APPswe: neuro-2 A neuroblastoma cells expressing human APP carrying the K670N/M671L Swedish mutation; Pen-2: presenilin enhancer 2; PS1: presenilin1.

Article Snippet: N2A cells stably expressing human APP carrying the K670N/M671L Swedish mutation (APPswe) were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin (Cellgro, Herdon, VA, USA), and 400 μg/mL G418 (Invitrogen), at 37°C in the presence of 5% CO 2 as previously described [ ].

Techniques: In Vitro, Incubation, Concentration Assay, Sandwich ELISA, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Mutagenesis

Journal: Journal of Neuroinflammation

Article Title: Involvement of 5-lipoxygenase activating protein in the amyloidotic phenotype of an Alzheimer’s disease mouse model

doi: 10.1186/1742-2094-9-127

Figure Lengend Snippet: In vitro effect of MK-591 on cAMP response element-binding protein and Notch. N2A-APPswe cells were incubated with increasing concentration of MK-591 or vehicle for 24 hours, and cell lysates collected for immunoassays. (A) Representative western blots of total CREB, its phosphorylated form (p-CREB) at Ser133, and Sp1 levels. (B) Densitometric analyses of the immunoreactivities to the antibodies to CREB, p-CREB and Sp1 (* P <0.01). (C) Representative western blots of NICD in the lysates of cells treated with MK-591, L685,458 or vehicle groups. (D) Densitometric analyses of the immunoreactivities to the antibodies NICD (n = 4; * P <0.01). CREB: cAMP response element-binding protein; NICD: Notch intracellular domain; N2A-APPswe: neuro-2 A neuroblastoma cells expressing human APP carrying the K670N/M671L Swedish mutation.

Article Snippet: N2A cells stably expressing human APP carrying the K670N/M671L Swedish mutation (APPswe) were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin (Cellgro, Herdon, VA, USA), and 400 μg/mL G418 (Invitrogen), at 37°C in the presence of 5% CO 2 as previously described [ ].

Techniques: In Vitro, Binding Assay, Incubation, Concentration Assay, Western Blot, Expressing, Mutagenesis

Journal: Genes & Diseases

Article Title: E674Q (Shanghai APP mutant), a novel amyloid precursor protein mutation, in familial late-onset Alzheimer's disease

doi: 10.1016/j.gendis.2023.02.051

Figure Lengend Snippet: The E674Q mutation facilitates the fragmentation of APP by BACE1 in vitro and in vivo . (A) Western blot analysis of HEK293 cells transfected with wild-type (WT) APP, APP E674Q, and APP with the Swedish mutation. (B, C) The levels of C99 and C83 fragments were increased in cells transfected with APP E674Q and APPswe. (D, H) ELISA quantification of the concentrations of Aβ40 (D) and Aβ42 (H) in conditioned medium from HEK293 cells transfected with wild-type and mutant APP. Data are presented as mean ± S.E.M. P < 0.01, one-way ANOVA. (E) Western blot analysis of AAV-mediated injection mice with APPwt, APPE674Q, and APPswe (Y188, ab32136, Abcam). (F, G) The levels of APP and C99 fragment were increased in mice hippocampus injected with AAV-APPE674Q and AAV-APPswe. (I–O) Behavioral experiments 6 months after the mice were injected. In the novel object recognition test, DR2 of APPE674Q animals was less than the control ( P < 0.05; J, K); in the Y maze test, APPswe ( P < 0.05) and APPE674Q ( P < 0.01) had a significant difference from the control but no noticeable difference from APPwt (L); in the Barnes maze test, the two APP mutation groups show a potentially faster learning trend than APPwt group (M, N); in the testing trail, APPswe ( P < 0.01) group had a noticeable shorter latency to escape than APPwt group (O).

Article Snippet: Adeno-associated virus vectors (AAVrh.9) encoding human mutant full-length APPswe, APPE674Q, APPwt, and empty control were purchased from Sangon Biotech, China.

Techniques: Mutagenesis, In Vitro, In Vivo, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Injection, Control