Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Article Title: Mouse brain proteomics establishes MDGA1 and CACHD1 as in vivo substrates of the Alzheimer protease BACE1.
doi: 10.1096/fj.201902347R
Figure Lengend Snippet: FIGURE 1 BACE1 substrate identification in vivo. A, Proteomic workflow of the Stable Isotope Labeling by Amino acids (SILA) spike-in approach. Isotopically heavy (13C6) labeled mouse brain tissue was combined with unlabeled (light) mouse brain tissue from wild-type (WT) or BACE1 KO mice. WT and BACE1 KO mice were littermates. B, Volcano plot of membrane fractions of BACE1 KO and WT mouse brains. The negative log10 transformed P value (y-axis) is plotted against the mean protein log2 fold change (x-axis) between BACE1 KO and WT samples. Single-span transmembrane proteins and GPI-anchored proteins are colored in blue. The hyperbolic curves represent a permutation-based FDR correction for multiple hypotheses. Proteins above the FDR curve remain significantly changed after FDR correction. C, Western blot analysis of the membrane preparation of WT, and BACE1 KO mouse brains at P3 shows accumulation of full-length CNTN2, CHL1, APP, MDGA1, and CACHD1. D, Statistical analysis was performed with n = 8 biological replicates using Mann-Whitney U test with the significance criteria of P < .05. All proteins accumulate significantly, except for CACHD1 that shows a nonsignificant trend to an increase. Graphs are presented with mean ± SEM
Article Snippet: Primary antibodies used: APP (Millipore, Merck; 22C11, dilution 1:1000), BACE1 (Robert Vassar, Northwestern University, Chicago, IL, USA; 3D5, dilution 1:1000), CHL1 (R&D Systems, Minneapolis, MN, USA; AF2147, dilution | 5NJAVRO et Al. 1:1000), CNTN2 (R&D Systems; AF4439, dilution 1:1000), β-actin (Sigma, Merck; AC-74, A5316, dilution 1:1000), Calnexin (Enzo Life Sciences, Germany; ADI-SPA-860, dilution 1:1000), HA (Covance, Princeton, NJ, USA; MMS101P, dilution 1:1000), NrCAM (Abcam, Cambridge, UK; ab24344, dilution 1:1000), and SEZ6 (dilution 1:10 of primary hybridoma supernatant).47 Monoclonal anti-CACHD1 antibody CACHI 15H10 (rat IgG1/k) (dilution 1:10 of primary hybridoma supernatant) was generated by immunization of Lou/c rats with the peptide NLENDRDERDDDSHEDR (intracellular part of murine and human CACHD1) using standard procedures.48 Monoclonal anti-MDGA1 antibody DGA 21E3 (rat IgG2a/k) (dilution 1:10 of primary hybridoma supernatant) was generated by immunization of Lou/c rats with native recombinant, C-terminally BAP-HIS-tagged murine MDGA1 ectodomain spanning amino acids 20-925 which was lentivirally transduced and overexpressed in HEK293T via a Gal4-UAS expression system and purified via metal affinity chromatography.
Techniques: In Vivo, Quantitative Proteomics, Labeling, Membrane, Transformation Assay, Western Blot, MANN-WHITNEY
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