Fig 2: ARL6IP1 mediates CNP-induced amelioration of AD-like pathologies in APP/PS1 mice. (A) Representative western blots (Left) and quantification (Right) of BACE1 in SH-SY5Y cells transiently transfected with siARL or scrambled (siNC), respectively. Antibody information: BACE1 (Abcam, ab108394), ARL6IP1 (Abcam, ab24228). (B) Aß40 and Aß42 levels in cell lysates (intracellular, in) and culture medium (extracellular, ex) were measured by ELISA from SH-SY5Y cells transiently transfected with siARL or siNC, respectively. (C) Relative luciferase activity of the 5'UTR of BACE1 in SH-SY5Y cells transiently transfected with siNC or siARL for 24 h, in the absence (CTRL) or presence of CNP (100 ng/mL) for further 24 h. (D) Representative western blots (Top) and quantification (Bottom) of BACE1 in SH-SY5Y cells transiently transfected with siARL or siNC for 24 h followed by CNP (100 ng/mL) or DMSO (CTRL) incubation for further 24 h. Antibody information: BACE1 (Abcam, ab108394), ARL6IP1 (Abcam, ab24228). (E–J) The hippocampus of male APP/PS1 mice (6 mo old) was bilaterally injected with AAV-shARL6IP1 (shARL) or AAV-shNC, and 4 wk later, mice were i.p. injected with vehicle (normal saline, NS) or CNP (10 µg/kg, every other day) for 2 more months, leading to the following four groups (9 mo old): shNC-NS, shNC-CNP, shARL-NS, and shARL-CNP, respectively. (E) Representative western blots (Top) and quantification (Bottom) of BACE1, sAPPß, ß-CTF, and ARL6IP1(ARL) levels in the hippocampus. Antibody information: BACE1 (Abcam, ab108394), sAPPß (BioLegend, SIG-39138), ß-CTF (IBL, 2B3), ARL6IP1 (Santa Cruz Biotechnology, sc-514476). (F) Soluble and insoluble Aß40 and Aß42 species were measured by ELISA in the hippocampus. (G) Immunofluorescent labeling (Left) and quantification (Right) of Aß (green) deposits. DAPI: nucleic marker (blue). (Scale bar: 200 µm.) Aß antibody: 4G8, BioLegend. (H) Representative images (Left) and quantification (Right) of Fluoro-Jade C (FJC, green)-stained cells in the hippocampus. (Scale bar: 50 µm.) (I) Representative images (Left) and quantification (Right) of TUNEL staining in the hippocampus. (Scale bar: 100 µm.) (J) Immunofluorescent labeling (Left) and quantification (Right) of IBA1 (microglia, green) and GFAP (astrocytes, red). (Scale bar: 200 µm.) Antibody information: IBA1 (GeneTex, GTX632426), GFAP (Proteintech, 60190-1-Ig). Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ns: nonsignificant.