Journal: The Journal of Biological Chemistry

Article Title: Familial Alzheimer’s disease mutations in amyloid precursor protein impair calcineurin signaling to NMDA receptors

doi: 10.1016/j.jbc.2024.108147

Figure Lengend Snippet: APP family members bind calcineurin. A , schematic overview of the orientation of APP in the plasma membrane ( gray lines ) and the relative location of β- and γ-secretase cleavage sites yielding the Aβ peptide ( red ). B , expanded view of the region surrounding the Aβ42 ( red ) sequence of APP. The CaN-binding site is highlighted ( yellow ). C , amino acid sequence similarity between the PVIVIT motif peptide and the corresponding CaN-binding sites in APP family members ( black background denotes identical; gray background denotes similar; white background denotes similarity with APP family members). D – F , representative western blots of co-IP experiments from rat brain extracts. Extracts (Ex) were subjected to immunoprecipitation (IP) with anti-CaN antibodies or a nonspecific IgG and immunoblotted (IB) for APP, APLP1, and APLP2 in panels D – F , respectively ( upper panels ). 5% of input was run in the extract lanes. Blots were stripped and reprobed with an anti-CaN antibody ( lower panels ). For validation of antibodies, see Fig. S1 . G , representative co-IP experiments from HEK 293 cells transfected with myc-His-CaN and Flag-tagged versions of APP family members. Extracts were IP’d with anti-Flag antibodies and probed with anti-His antibodies ( upper panel ). Blots were stripped and reprobed with anti-Flag antibodies. H , in vitro interaction of recombinant GST-CaN (200 ng) with the indicated biotinylated peptides (10 μg). Peptides were precoupled to streptavidin-coated beads and incubated with GST-CaN (200 ng). Immunoblots were performed using anti-CaN antibodies. A representative blot is shown ( upper panel ). Summary bar graph from multiple experiments in shown ( lower panel ). Data are normalized to the GST-CaN standard (80 ng) and adjusted for the percentage of input represented by the standard (40%) and expressed as mean ± s.e.m. Overlaid individual data points are from biological replicates. Ht31 is a nonspecific control peptide, and beads represent a beads alone control. ∗ p < 0.05; ∗∗ p < 0.01 compared to PVIVIT ( black ) and Ht31 ( red ) evaluated by a one-way ANOVA (F (6,38) = 32.050; p = 2.02 × 10 -13 ∗∗) and Tukey’s post hoc test.

Article Snippet: Recombinant rabbit monoclonal to APP (clone Y188; Abcam cat# ab32136; 1:5000 dilution), rabbit polyclonal to APLP1 (Proteintech cat # 12305-2-AP; 1:1000 dilution), and rabbit polyclonal to APLP2 (Proteintech cat # 15041-1-A; 1:500 dilution) were used to detect native APP family members on western blots or recombinant GFP-tagged APP family members as validation (see ).

Techniques: Clinical Proteomics, Membrane, Sequencing, Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Biomarker Discovery, Transfection, In Vitro, Recombinant, Incubation, Control

Journal: medRxiv

Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy

doi: 10.1101/2024.08.17.24311658

Figure Lengend Snippet: SNX17-Retriever/CCC/WASH pathway is essential for recycling of Reelin signaling receptors, APP family, and SLITRK family proteins. (A) Representative blots of co-expression of mCherry-tagged SNX17 with GFP-tagged cytoplasmic tail of LRP8 and VLDLR from three independent experiments. The interactions between mCherry-SNX17 and wild-type GFP-tagged cytoplasmic tails of Human-LRP8 and Human-VLDLR were significantly decreased when NPxY was substituted with NPxA. (B) Representative blots of analysis from three independent experiments using DIV17 rat cortical neurons transduced with either a scramble-control, SNX17, or VPS35L shRNA. Bar graphs show relative protein abundance of LRP8 and VLDLR in cell lysate or cell surface. (C) DIV17 rat cortical neurons transduced with shRNA were incubated for 30 min with or without AP-Reelin, followed by cell lysis and western blot analysis. Bar graph shows quantification of band intensities of AP relative to cells transduced with sh-SCR control from three independent experiments. (D) DIV17 rat cortical neurons transduced with shRNA were incubated for 7 min with or without Reelin. Phosphorylation level of Dab1 was then measured using immunoprecipitation and western blot analysis. Representative blots and quantification from three independent analyses are shown. (E) Volcano plots of transmembrane proteins with decreased (blue circles) or increased (red circles) cell surface abundance in sh-VPS35L suppression comparedto sh-SCR in DIV17 rat cortical neuron from three independent TMT-based proteomic experiments. Two independent sh-RNAs for VPS35L were used to avoid off-target effects. (F) Enrichment analysis of significantly downregulated proteins (LogFC < −0.32, p < 0.05) in the sh-VPS35L compared to sh-SCR by Metascape. (G) Representative blots of mCherry-nanotrap for mCherry-SNX17 under co-overexpression of chimeric constructs of Human-SLITRK family proteins from three independent experiments. All proteins except for SLITRK4 have an NPxY motif, which was mutated to NPxA in the mutant. (H) Representative blots of mCherry-nanotrap for mCherry-SNX17 under co-overexpression of chimeric constructs of Human-APP family proteins from three independent experiments. APP, APLP1, and APLP2 have NPxY motifs, and the NPxY motif was mutated to NPxA in the mutant. (I) Representative blots for APP and APLP2 in rat cortical neurons. Cell surface protein fraction was obtained, and N-Cadherin were used for loading control. Bar graphs show relative values of cells with sh-SNX17 or sh-VPS35L suppression compared to sh-SCR control cells (n=3). In all graphs, error bars represent mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: The following antibodies were used in this study (WB: western blot, IF: immunofluorescence): rabbit anti-SNX17 (Proteintech, 10275-1-AP, WB), mouse anti-GFP (Roche, 11814460001, WB), rabbit anti-GFP (GeneTex, GTX30738, WB), mouse anti-mCherry (antibodies.com, A85305, WB), rabbit anti-mCherry (antibodies.com, A85306, WB), rabbit anti-CCDC22, (Proteintech, 16636-1-AP, WB), mouse anti-CCDC93, (Origene, CF800568, WB), rabbit anti COMMD4 and rabbit anti COMMD9 (kind gift from Prof. Ezra Burstein, WB) rabbit anti-C16orf62 (Abcam, ab97889, WB), rabbit anti-C16orf62 (Pierce, PA5-28553, IF), rabbit anti-DSCR3 (Merck Millipore, ABN87, WB), rabbit anti-integrin-β1 (Abcam, ab52971, WB), goat anti-VPS35 (antibodies.com, A83699, IF), mouse anti-VPS29 (Santa Cruz, sc-398874, WB), rabbit anti-KIAA1033 (Proteintech, 51101-1-AP, WB), mouse anti-Strumpellin (Santa Cruz, sc-377146, WB), mouse anti-β actin (Sigma, A1978, WB), rabbit anti-LRP1 (Abcam, ab92544, WB), rabbit anti-LRP2 (Proteintech, 19700-1-AP, WB), rabbit anti-LRP2 (prepared as described in , IHC), rabbit anti-LRP4 (SIGMA, HPA012300, WB), rabbit anti-LRP8 (Abcam, ab108208, WB), mouse anti-VLDLR (Santa Cruz, sc-18824, WB), rabbit anti-APP (Abcam, ab32136, WB), rabbit anti-APLP2 (Proteintech, 15041-1-AP, WB), mouse anti-AP (Thermo Fisher, MA1-20245, WB), rabbit anti-Dab1 (kind gift from Dr. M Hattori, IP/WB) , mouse anti-Phosphotyrosine (Merck, 05-321, WB), rabbit anti-GLUT1 (Abcam, ab115730, IF/WB), mouse anti-N-cadherin (Cell signalling technology, 14215S, WB), anti-PSD95 (Merck, MAB1596, WB), mouse anti-FLAG (SIGMA, F1804, WB), rabbit anti-FGFR2, (Proteintech, 13042-1-AP, WB), rabbit anti-ERK1/2 (Cell Signaling Technology, 9102, WB), rabbit anti-phospho-ERK1/2 (Cell Signaling Technology, 9101, WB), anti Ctip2 (Abcam, ab18465, IHC), anti Tbr1 (Abcam, ab275960, IHC), anti Neun (Cell signalling technology, 12943, IHC).

Techniques: Expressing, Transduction, Control, shRNA, Incubation, Lysis, Western Blot, Immunoprecipitation, Over Expression, Construct, Mutagenesis