Fig 1: 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.
Fig 2: CNP promotes ARL6IP1–FXR1 interaction but inhibits FXR1–5'UTR binding in human cells. (A) Western blots show FXR1 that is coimmunoprecipitated with ARL6IP1 (ARL, Top), or ARL that is coimmunoprecipitated with FXR1 (Middle) is enhanced in SH-SY5Y cells treated with CNP (100 ng/mL) for 24 h, whereas the protein level of ARL and FXR1 in input is not significantly altered (Bottom). Antibody information: FXR1 (Abcam, ab129089), ARL6IP1 (Abcam, ab24228). (B) Immunofluorescent images show the colocalization of ARL with FXR1 (i) or FXR2 (ii) in SH-SY5Y cells treated with CNP (100 ng/mL) or DMSO (CTRL) for 24 h. (Scale bar: 25 µm.) PCC: Pearson’s correlation coefficient. Antibody information: ARL6IP1 (GeneTex, GTX85516), FXR1 (Santa Cruz Biotechnology, sc-374148), FXR2 (Santa Cruz Biotechnology, sc-32266). (C) Immunofluorescent images of Venus (green, autofluorescence) and FXR1 (red) in SH-SY5Y cells cotransfected with BACE1 5'UTR-8xCBS and VN-dEcCas6-VC plasmid for 48 h. (Scale bar: 10 µm.) FXR1 antibody: Santa Cruz Biotechnology, sc-374148. (D) Representative immunofluorescent images (Left) and quantification (Right) of the colocalization between FXR1 (red) and the 5'UTR-labeled GFP (green, autofluorescence), in SH-SY5Y cells cotransfected with MS2-BACE1 5'UTR and MCB-GFP plasmid for 24 h, with CNP (100 ng/mL) or DMSO (CTRL) treatment for further 24 h. (Scale bar: 10 µm.) FXR1 antibody: Santa Cruz Biotechnology, sc-374148. (E) Relative BACE1 mRNA levels measured by RT-qPCR in cell extracts immunoprecipitated by FXR1 or IgG antibody. SH-SY5Y cells were treated with CNP (100 ng/mL) or DMSO (CTRL) for 24 h. Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01. ns: nonsignificant.
Fig 3: ARL6IP1 interacts with the 5'UTR-targeted RBP FXR1. (A) Schematic diagram for the RNA pulldown combined with MS assay. 5-bromo-UTP (BrU) was randomly incorporated into the 5'UTR upon in vitro transcription. The BrU-labeled 5'UTR was mixed with cell lysates and pulled-down by BrU antibody for subsequent MS analysis. (B) The BACE1 5'UTR-targeted RBPs FXR1, FXR2, and DDX17 are interacted with each other, analyzed by online STRING software. (C) Representative western blots of FXR1, FXR2, DDX17, and GAPDH in immunoprecipitated extracts by RNA (BACE1 5'UTR) pull-down assay in SH-SY5Y cells. Antibody information: FXR1 (Abcam, ab129089), FXR2 (Proteintech, 12552-1-AP), DDX17 (Abcam, ab180190), GAPDH (Proteintech, 60004-1-Ig). (D) Representative western blots of FXR1, FXR2, and DDX17 in immunoprecipitated extracts by ARL6IP1(ARL) antibody in SH-SY5Y cells. Antibody information: FXR1 (Abcam, ab129089), FXR2 (Proteintech, 12552-1-AP), DDX17 (Abcam, ab180190), ARL6IP1 (Abcam, ab24228). (E) Representative western blots of FXR1, FXR2, and FMR1 in immunoprecipitated extracts by flag antibody in SH-SY5Y cells transiently transfected with the 3×flag-ARL6IP1 plasmid for 72 h. Antibody information: FXR1 (Abcam, ab129089), FXR2 (Proteintech, 12552-1-AP), FMR1 (Abcam, ab259335), ARL6IP1 (Abcam, ab24228). (F) Representative western blots of BACE1 (i) and FXR2 (ii), and the quantification (Right) in SH-SY5Y cells transfected with FXR2 siRNA (siFXR2) or siNC for 24 h, followed by CNP (100 ng/mL) or DMSO (CTRL) treatment for further 24 h. Antibody information: BACE1 (Abcam, ab108394), FXR2 (Proteintech, 12552-1-AP). (G) Representative western blots of BACE1 (i) and FXR1 (ii), and the quantification (Right) in SH-SY5Y cells transfected with FXR1 siRNA (siFXR1) or siNC for 24 h, followed by CNP (100 ng/mL) or DMSO (CTRL) treatment for further 24 h. Antibody information: BACE1 (Abcam, ab108394), FXR1 (Abcam, ab129089). (H) Relative BACE1 5'UTR-luciferace activity in SH-SY5Y cells transfected with siFXR1 or siNC for 24 h, followed by CNP (100 ng/mL) or DMSO (CTRL) for 24 h. Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ns: nonsignificant.
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