Human fresh tissues were fixed in a 4% paraformaldehyde solution. Tissue sections (5 µm) were treated with target retrieval buffer (Dako, Carpinteria, CA, USA) followed by endogenous peroxydase activity blockade with 3% hydrogen peroxide (Merck, Darmstadt, Germany). Non-specific binding sites were blocked with universal blocker (BioGenex, Fremont, CA, USA). Rabbit anti-ADAM10 antibody (ab1997, Abcam, Cambridge, UK, 1/1000) was incubated 2 h at room temperature (RT) followed by incubation with a swine anti-rabbit horseradish peroxidase (HRP)-linked antibody (Dako, 1/3000) for 30 min at RT. Sections were then stained with 3,3′ diaminobenzidine tetrachloride (DAB) (Dako) and digitalized using NanoZoomer 2.0-HT system (0.23 µm/pixel (×40) scanning resolution). Quantification of ADAM10 staining in each sample was based on a score of staining intensity ranging from 0 (absence of staining) to 3 (strong staining).
Ab1997
Manufactured by Abcam
Sourced in United Kingdom
Ab1997 is a laboratory equipment product. It is designed to perform a core function within the scientific research and testing process. The detailed specifications and intended use of this product are not available for an unbiased and factual presentation.
Automatically generated - may contain errors
Lab products found in correlation
Ab2077, by Abcam (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Target retrieval buffer, by Agilent Technologies (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
A8717, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Abcam (1 mentions)
Sapp β sig 39138, by BioLegend (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ab84036, by Abcam (1 mentions)
Zen black software, by Zeiss (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Ab216130, by Abcam (1 mentions)
Ab652, by Abcam (1 mentions)
Trupage precast gels, by Merck Group (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Ab53554, by Abcam (1 mentions)
Ab15690, by Abcam (1 mentions)
Nel105001ea, by PerkinElmer (1 mentions)
Anti adam10, by Abcam (1 mentions)
8 protocols using ab1997
1
Immunohistochemical Analysis of ADAM10 Expression
2
Sulfuretin-Modulated Regulation of APP Processing
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Sulfuretin was purchased from Xili (Kunming, Yunnan, China), dimethyl sulfoxide (DMSO) was purchased from Dinguo (Beijing, China). Actinomycin D (ActD), cycloheximide (CHX), chloroquine (CQ), PKA inhibitor H89, mTOR inhibitor rapamycin (RAPA), PPAR-α inhibitor GW6471, ERK inhibitor U0126, β-secretase inhibitor LY2811376, α-secretase inhibitor GI254023X, γ-secretase inhibitor DAPT, PKA inhibitor H89 and PI3K inhibitor LY429002, 3-MA were purchased from MCE. All chemicals were dissolved in DMSO to generate a 20 mM stock solution. Subsequent dilutions were performed by culture medium. The final concentration of DMSO was no more than 1:1000.
Antibody information was as follows: ADAM10 (ab1997, Abcam, 1:1000); BACE1 (ab2077, Abcam, 1:1000); APP-full length (A8717, Sigma, 1:3000); α-CTF and/or β-CTF: A8717 (Sigma; 1:3000), 4G8 (BioLegend, 1:1000) and 6E10 (BioLegend, 1:1000); SP1 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500); USF1 (Santa Cruz Biotechnology, 1:500); RXR-α (Santa Cruz Biotechnology, 1:500); sAPPα (2B3, IBL, 1:500); sAPPβ (SIG-39138, Biolegend, 1:500); GAPDH (Proteintech, 1:10000). Horseradish peroxidase-conjugated anti-rabbit, anti-mouse and anti-goat secondary antibodies were purchased from Proteintech (Wuhan, China).
Antibody information was as follows: ADAM10 (ab1997, Abcam, 1:1000); BACE1 (ab2077, Abcam, 1:1000); APP-full length (A8717, Sigma, 1:3000); α-CTF and/or β-CTF: A8717 (Sigma; 1:3000), 4G8 (BioLegend, 1:1000) and 6E10 (BioLegend, 1:1000); SP1 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500); USF1 (Santa Cruz Biotechnology, 1:500); RXR-α (Santa Cruz Biotechnology, 1:500); sAPPα (2B3, IBL, 1:500); sAPPβ (SIG-39138, Biolegend, 1:500); GAPDH (Proteintech, 1:10000). Horseradish peroxidase-conjugated anti-rabbit, anti-mouse and anti-goat secondary antibodies were purchased from Proteintech (Wuhan, China).
3
Immunocytochemistry: Quantifying Protein Colocalization
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For immunocytochemistry, cells were fixed with 4% PFA + 4% sucrose followed by blocking and permeabilization with 2% normal donkey serum plus 0.1% saponin for 1 h. Primary antibodies targeting either A10 (1:200; ab1997, rabbit; Abcam), PS1 (1:1,000; 13A11, mouse), or TFR (both 1:200; ab84036, rabbit [Abcam]; H68.4, mouse [Thermo Fisher Scientific]) were incubated overnight at 4°C in blocking buffer. The following day, cells were washed with PBS and treated with secondary antibody for 1 h at a dilution of 1:2,000 in PBS followed by washes with PBS. Hoechst was included in the second to last wash at a dilution of 1:2,000. All slides were mounted with Vectashield mounting medium (Vector Laboratories). 1-μm optical sections were acquired on a microscope (LSM-710; Carl Zeiss) using a 63× oil immersion objective. All images were processed and analyzed with Zen Black software (Carl Zeiss). To quantify colocalization, all images were stained together in the same session along with control samples where one or both primary antibodies were omitted (to calculate background fluorescence). Automated colocalization analysis of at least 10 images (∼40 cells) was done with Zen Black software according to documented protocols.
4
Exosomal Protein Analysis by Western Blot
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About 10 µg of exosomal proteins were lysed with Laemmli sample buffer (Cat. # 1610747) prior to loading onto a 10% TruPAGE™ Precast Gels (Cat. # PCG2001, Sigma-Aldrich). The resolved proteins were transferred to a nitrocellulose membrane (Cat. #1620167, Bio-Rad) using Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in 1X TBS 1% casein blocker for 2 h at room temperature and incubated with primary antibody (1:1,000 dilution) overnight at 4 °C with slow shaking. The membrane was probed against rabbit anti-ADAM10 antibody (ab1997, Abcam), anti-glypican 1 (GPC-1) antibody (ab199343, Abcam), anti-glucose transporter (GLUT-1) antibody (ab652, Abcam), anti-CD63 antibody (ab216130, Abcam) and CD81 recombinant rabbit monoclonal antibody (SN206-01, ThermoFisher Scientific). Membranes were washed three times in 1 × TBST buffer and incubated with a goat anti-rabbit IgG HPR-conjugated secondary antibody (1:5,000 dilution) for 2 h at room temperature with shaking. Pierce ECL Western Blotting substrate (catalog no. 32209, Thermo Scientific) was used to visualize specific antigen–antibody binding.
5
Hippocampal Protein Expression Analysis
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The hippocampi were homogenized in a pro-prep solution (17081, iNtRon, Gyeonggi-do, Korea) after physically mincing. The protein concentration was quantified using the Bradford reagent (Bio-Rad, Hercules, CA, USA), and then denatured by boiling for 5 min, depending on the concentration of the sample. Each sample was separated by electrophoresis on 10–15% sodium dodecyl sulfate-polyacrylamide gels. Then, the proteins were transferred to a nitrocellulose membrane and blocked for 30 min with 5% bovine serum albumin (BSA) solution for nonspecific blocking. Membranes were incubated at 4 °C with anti-6E10 (1:1000, Sig-39320, Covance), anti-ADAM10 (1:1000, ab1997, Abcam, Cambridge, UK), anti-BACE (1:1000, sc-33711, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GFAP (1:1000, ab53554, Abcam), anti-Iba1 (1:1000, ab15690, Abcam), or anti-β-actin (1:3000, #3700, Cell Signaling, Danvers, MA, USA). After overnight incubation, membranes were washed thoroughly in PBS-T buffer and incubated for 1 h with the corresponding secondary antibody diluted at 1:3000. The membranes were then washed, and immunoreactivity was detected using an enhanced chemiluminescence (ECL) kit (NEL105001EA, Perkin Elmer, Waltham, MA, USA).
6
Western Blot Analysis of ADAM10 and ADAM17
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The cells were lysed in ice-cold RIPA buffer enriched with 5 mM EDTA, Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific), and 5 μM ADAM10-specific inhibitor GI254023X (Brummer et al., 2018 (link)). After brief sonication, protein samples (25 μg) were subjected to tris-glycine SDS-PAGE and transferred onto 0.45 μm PVDF membranes (Immobilon P, Merck). The membranes were stained with Ponceau S to ensure equal protein loading and images were taken. The membranes were destained, blocked in 5% non-fat dried milk in TBST and probed with rabbit-anti ADAM17 at 1:2500 (PA5-17080, Thermo Fisher Scientific) or rabbit anti-ADAM10 at 1:5000 (ab1997, Abcam) and then with HRP-conjugated secondary antibody (goat anti-rabbit at 1:10,000, Sigma-Aldrich). Bands were developed with Immobilon Western Chemiluminescent HRP Substrate (Merck) and visualized using Fusion FX (Vilber Lourmat). The exposition time was set to ‘auto’.
7
Quantitative Analysis of Alzheimer's Biomarkers
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Equal amounts of TPER-soluble protein (20 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS□PAGE) on a 4–12% Bis/Tris gel (Bio-Rad, Hercules, CA), transferred to 0.20 μm nitrocellulose membranes, blocked for 1 h in 5% (vol/vol) nonfat milk or bovine serum albumin (BSA) in Tris□buffered saline (pH 7.5) supplemented with 0.2% Tween20. Antibodies and dilutions used include anti-6E10 (6E10; 1:1000; 803001; BioLegend), anti-APP C-Terminal (APP; 1:1000; 171610; Calbiochem, San Diego, CA) for C99 and C83, anti-ADAM10 (1:1000; ab1997; Abcam), anti-β-secretase 1 (BACE1; 1:1000; ab2077; Abcam), anti-Presenilin-1 (PS1; 1:1000; ab76083; Abcam), anti-Presenilin enhancer 2 (PEN2; 1:500; ab18189; Abcam), anti-A11 (A11; 1:1000; AHB0052; Thermo-Fisher Scientific), and anti-β-actin (1:10,000; MA5–15739; Sigma-Aldrich). Blots were developed using Pico/Dura Western Blotting Detection System (Pierce) and exposed to film for images. Quantitative densitometric analyses were performed on digitized images of immunoblots using the NIH program ImageJ and band densities were normalized to β-actin. Uncropped scans of the blots are presented in Supplementary Figure 10A and 10B and provided in Source Data file.
8
Proteomic Analysis of Apoptosis and EMT Regulators
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Cells were lysed in radio-immunoprecipitation assay cell lysis buffer (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) to collect the total protein of cells. The protein concentration was measured using a bicinchoninic acid method. Then, the protein was run on SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Following 2 h of 5% bovine serum albumin treatment to block non-specific binding, the membranes were incubated with the primary antibodies to B-cell lymphoma-2 (Bcl-2, 1:1000, ab32124, Abcam) and Bcl-2-associated X (Bax, 1:1000, ab32503, Abcam), ADAM10 (1:1000, ab1997, Abcam), N-cadherin (1:1000, ab18203, Abcam), matrix metalloproteinase-9 (MMP-9, #13,667, Cell Signaling Technology, Boston, UK), Signal transducer and activator of transcription 3 (STAT3, 1:1000, #30,835, CST), p-STAT3 (1:2000, #9145, CST), GAPDH (1:2000, ab9485, Abcam) at 4°C overnight, and then incubated with goat-anti rabbit secondary antibody (ab205719, 1:2000, Abcam) at 20°C for 1 h. After that, the protein blots were developed using enhanced chemiluminescence reagent (EMD Millipore, Corp. Billerica, MA, USA), and the grey value was determined using an Image J software. Three independent experiments were performed.
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