Cell or mouse muscle and brain extracts were prepared in lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, halt proteinase inhibitor cocktail (ThermoFisher Scientific) and halt phosphatase inhibitor cocktail (ThermoFisher Scientific), and subjected to western blot analysis with anti-LC3 (Novus Biologicals, NB100-2220), anti-SQSTM1 (Abnova, H00008878-M01), anti-Aβ42 (Invitrogen; 700254), anti-APP (Biolegend; 803001), HRP-conjugated GFP antibody (Santa Cruz Biotechnology, sc9996), anti-HA (Cell Signaling Technology, C29F4), anti-ATG7 (Sigma Aldrich, A2856), anti-LDLR (Abcam, ab52818), anti-LRP1 (Abcam, ab92544), and anti-ACTB/β-actin-HRP (Santa Cruz Biotechnology, sc47778 HRP) antibodies. The band intensity was analyzed using the ImageJ software.
Anti atg7
Manufactured by Merck Group
Sourced in United States
Anti-Atg7 is a laboratory reagent used in research applications. It functions as an inhibitor of the autophagy-related protein 7 (Atg7), which plays a crucial role in the autophagy process. The core function of Anti-Atg7 is to modulate the activity of Atg7 in experimental settings, allowing researchers to study the impact of Atg7 inhibition on cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti p62, by Abcam (2 mentions)
Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Anti ldlr, by Abcam (1 mentions)
Anti lc3, by Novus Biologicals (1 mentions)
Anti aβ42, by Thermo Fisher Scientific (1 mentions)
Anti lrp 1, by Abcam (1 mentions)
Halt proteinase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated gfp antibody, by Santa Cruz Biotechnology (1 mentions)
Anti app, by BioLegend (1 mentions)
Anti sqstm1, by Abnova (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Monoclonal anti c myc antibody, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Cell counting kit, by Yeasen (1 mentions)
6 protocols using anti atg7
1
Western blot analysis of autophagy and Alzheimer's markers
2
Apoptosis and Autophagy Analysis
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DMEM (Gibco, C11965500BT), fetal bovine serum (Gibco, 16000044), EBSS (MACGENE, CC026), FITC Annexin V apoptosis detection kit I (BD Biosciences, 556547), cell counting kit (YEASEN, QF0026), lysis buffer (Beyotime, P0013), PVDF membranes (Millipore, ISEQ00010), albumin bovine V (Solarbio, A8020), lipofectamine 2000 (Invitrogen, 11668027), Dual-Luciferase® Reporter Assay System (Promega, E1910), and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining kit (Promega, G3250). Bafilomycin A1 was purchased from Sigma. The following antibodies were used: anti-ULK1 (Sigma-Aldrich, A7481), anti-P62 (MBL, PM045), anti-P62 (Abcam, ab56416), anti-LC3B (Sigma, L7543), anti-c-Myc monoclonal antibody (Sigma, C3956), anti-β-actin (Sino Biological, 100162-RP02-100), anti-Gapdh (Transgen, HC301), anti-ULK1 (Sigma, A7481), anti-ATG5 (Sigma, A0731), anti-ATG7 (Sigma, A2856), anti-Beclin1 (CST, 3738), anti-Tom20 (BD Biosciences, 612278), DAPI (CST, 4083), HRP Affinipure goat anti-mouse IgG (Earthox, E030110), and HRP Affinipure goat anti-rabbit IgG (Earthox, E030120). The following fluorescent secondary antibodies were used: Alexa Fluor 555-labeled donkey anti-mouse IgG antibodies (Invitrogen, A31570) and Alexa Fluor 488-labeled donkey anti-rabbit IgG antibodies (Invitrogen, A21206).
3
Western Blot Analysis of Autophagy and Apoptosis
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The protein samples were prepared with RIPA lysis buffer supplemented with protease inhibitor and phosphatase inhibitor mix and boiled at 95°C for 10 min. The proteins were separated by 10% SDS gel and transferred to polyvinylidene fluoride (PVDF) membranes in a cold room. After that, the PVDF membranes were blocked with 5% skimmed milk [dissolved in Tris-buffered saline-Tween (TBST)] at room temperature (37°C) for 2 h and then incubated overnight with a primary antibody with gentle shaking at 4°C. After five washes with TBST, the membrane was incubated with a peroxidase-conjugated secondary antibody at room temperature (37°C) for 2 h. Following five washes with TBST, the target protein was visualized with chemiluminescence (ECL) (Thermo Fisher Scientific, Beijing, China). The primary antibodies used in this study include anti-LC3-I/II, anti-p62, anti-bECLin-1, and anti-Atg7 (Sigma-Aldrich, St. Louis, MO, USA) and anti-cleaved caspase 3 (Abcam, Cambridge, UK). GAPDH (Abcam) was used as an internal control.
4
Autophagy Protein Quantification in Placenta
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Total protein was extracted form frozen human and mouse placenta samples and homogenized in RIPA buffer (Cell Signaling Technology) with protease inhibitor cocktails (Sigma-Aldrich). Equivalent amounts of total protein, determined by BCA assays (Thermo Scientific), were separated on 4%–20% Mini-PROTEAN precast gels (Bio-Rad) and transferred to PVDF membranes. The following primary antibodies and dilutions were used: anti-LC3B (1:1,000; Novus, NB600-1384), anti-P62 (1:1,000; Abcam, ab56416), anti-Atg7 (1:1,000; Sigma-Aldrich, A2856), anti-Atg16L1 (1:1,000; Sigma-Aldrich, A7356), anti-Beclin1 (1:1,000; Cell Signaling, 3738s), and anti-Actin (1:2,000; Cell Signaling, 3700s). ImageJ (NIH) was used for densitometry of Western blots.
5
Quantifying Autophagy-related Proteins
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Cell and tissue lysate were prepared as described previously [23 (link)] and quantified by BCA assay. Total protein (~20-30 μg) was loaded and resolved in a Novex 4-12% Bis-Tris gel (Life Technologies, Carlsbad, CA). Protein was transferred to an Immobilon-P PVDF membrane (Millipore, Billerica, MA) and probed with anti-LC3-II (Novus Biologicals, Littleton, CO), anti-Beclin-1 (Novus Biologicals, Littleton, CO), anti-Atg7 (Sigma, St. Louis, MO), anti-p62 (BioLegend, San Diego, CA) or anti-Keap1 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. Anti-ɑ–tubulin (Cell Signaling Technology, Danvers, MA) or anti-GAPDH (Imgenex, San Diego, CA) were used as loading controls. Protein levels were quantified using densitometric scanning and normalized to loading control levels.
6
Western Blotting of Autophagy and Apoptosis Proteins
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Cell lysates were prepared by RIPA buffer (1XPBS, 1% nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 8.0). Protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, US). After equal amounts of proteins were loaded to the gels, proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes (EMD Millipore, Thermo Fisher Scientific, US). Following the classic immunoblotting steps (blocking, incubating with primary and secondary antibodies), chemiluminescence signals were detected using Clarity ECL substrate solution (Bio-Rad, US) by Fusion-FX7 (Vilber Lourmat, Thermo Fisher Scientific, US). Monoclonal antibodies used in this study were anti-LC3 (CST-12741, USA), anti-Atg-5/12 (CST-12994, USA), anti-Atg-7 (CST-8558, US), anti-actin (Sigma-Aldrich-A5316, UK), anti-caspase-3 (CST-9665, US), Anti-CHOP (CST-2895, UK). The experiments were repeated three times independently; with one representative result shown.
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