Computational protocol: The Amyloid Precursor Protein Is a Conserved Receptor for Slit to Mediate Axon Guidance

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Protocol publication

[…] Full-length (FL) human APP695, APPΔE1, APPΔE2, and APPΔC expression constructs were described previously (). Human Slit2 tagged with c-Myc (Slit2-Myc) and rat Robo1 tagged with the hemagglutinin (Robo1-HA) were described previously (). Constructs containing Slit2 N-terminal (Slit2N) and C-terminal (Slit2C), according to , were generated using the Q5 Site-Directed Mutagenesis kit (New England Biolabs) and confirmed with sequencing. FL Slit2 fused N terminally to the human alkaline phosphatase sequence was generated from Ntn5.a-AP-His (Addgene) after excision of Netrin 5 from the vector. Other constructs used in this study include HA-Fe65 () and Myc-Pak1 (). pRK5-Myc-Rac1 were purchased from Addgene. The ADAM10 inhibitor GI254023X (Sigma) was described previously ().HEK293 cells were cultured in DMEM supplemented with 10% fetal bovine serum. For cell surface binding assay, HEK293 cells plated on glass coverslips were transiently transfected with either APP695, Robo1-HA or APP deletion constructs using the lipofectamine reagent (Invitrogen). Twenty-four hours later, the cells were incubated with serial dilutions of previously dosed supernatants from HEK293 cells expressing Slit2-Myc supplemented with 1 μg/ml heparin for 1.5 h at 37°C. After washing three times in PBS, cells were fixed in 4% PFA for 20 min at room temperature and permeabilized with 0.1% NP40 in PBS for 15 min. Double immunofluorescent labeling of transfected receptors and bound Slit2 were conducted using the anti-APP antibody (4G8, Biolegend), mouse HA antibody (6E2, Cell Signaling) and rabbit Myc antibody (A14, Santa Cruz Biotechnology) followed by incubation with secondary antibodies. For colocalization quantification, images were background-subtracted and region of interest of one cell was delineated. Quantitative colocalization analysis evaluating the average proportion of Slit2 signals colocalized with Robo, FL, or mutant APP was performed over the entire fluorescence images using the plugin ‘Coloc 2’ on the Fiji software (ImageJ). The thresholded Manders M1 coefficient was expressed and compared between groups.For quantitative binding assays, HEK293 cells were transfected with either FL APP or HA-Robo. Twenty-four hours later, the cells were incubated with serial dilutions of previously dosed supernatants from HEK293T cells expressing AP-tagged Slit2 and 1 μg/ml heparin for 2 h at 37°C. After washing with cold PBS, Alkaline Phosphatase activities in the cell lysates were measured with the Alkaline Phosphatase Assay kit (AbCam). Optical density at 405 nm was determined using a SpectraMax i3x microplate reader (Molecular Devices). Binding curves were fitted using the Hill equation, and corresponding dissociation constant (Kd) values were calculated with Prism 6 software (GraphPad).Cortical neurons from postnatal day 0 (P0) mouse were plated in six-well poly-D-lysine coated plates at 106 cells/well for 7 days in vitro (DIV). In the coculture assay, the HEK293 cell expressing Slit2 were seeded on cell culture inserts (Costar) and transferred to 6 DIV primary neurons. The conditioned media (CMs) and cell lysates were collected for Western blot analysis 24 h later. […]

Pipeline specifications

Software tools Coloc, ImageJ
Databases Addgene
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Caenorhabditis elegans
Diseases Alzheimer Disease