Following washing 3 times with ice-cold 1×PBS, the HK-2 cells were lysed in RIPA lysis buffer and total protein extracts was collected in centrifuge tubes. After centrifugation (13 000 rpm for 15 minutes at 4°C) protein concentration was determined using the bicinchoninic acid (BCA) reagent method. Then 40 μg of total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After that, the membranes were blocked in 5% non-fat milk for 1 hour at 37°C and then incubated overnight at 4°C with primary antibodies. Primary antibodies used in this study were: anti-Beclin-1 (1: 1000; Abcam); anti-LC-3II (1: 1000; Abcam); anti-p62 (1: 1000; Abcam); and anti-β-actin (1: 1000; Abcam). After washing 3 times for 15 minutes with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (1: 1000; Cell Signaling Technology, Inc.) for 2 hours at room temperature. Following development with Odyssey Fc System (LICOR, USA), Western blots were densitometrically analyzed using ImageJ software.
Anti lc3ii
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-LC3II is a primary antibody that specifically recognizes the LC3-II form of the microtubule-associated protein 1 light chain 3 (LC3). LC3-II is a marker of autophagosome formation and is widely used to monitor autophagic activity in cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti beclin1, by Abcam (4 mentions)
Anti p62, by Abcam (3 mentions)
Anti mtor, by Abcam (2 mentions)
Anti lc3i, by Abcam (2 mentions)
Anti tubulin, by Abcam (2 mentions)
Odyssey fc system, by LI COR (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anti β actin, by Abcam (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Anti sqstm1 p62, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Aβ1 42, by Thermo Fisher Scientific (1 mentions)
Anti parkin, by Abcam (1 mentions)
Anti synapsin 1, by Abcam (1 mentions)
Donepezil, by Santa Cruz Biotechnology (1 mentions)
Bicinchoninic acid bca protein assay kit, by Beyotime (1 mentions)
Anti pink1, by Abcam (1 mentions)
Immunol fluorescence staining kit, by Beyotime (1 mentions)
10 protocols using anti lc3ii
1
Quantification of Autophagy Proteins
2
Alzheimer's Disease Pathogenesis Model
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The Aβ1-42 used in these experiments was acquired from Life Technologies (USA); β-asarone was purchased from NIFDC (Beijing, China), and the purity value is 96.8%; donepezil was obtained from the First Affiliated Hospital of Jinan University (Guangzhou, China); A3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); CSA was purchased from Selleck (Houston, Texas, USA); 3MA, high-glucose DMEM, FBS, trypsin, and PBS were obtained from Gibco (Gaithersburg, MD, USA); Cell Counting Kit-8 (CCK-8), Cytotoxicity LDH Assay Kit—WST (LDH), and Cellular Senescence Detection Kit—SPiDER-βGal were obtained from Dojindo Molecular Technologies, Inc. (Tokyo, Japan); the bicinchoninic acid (BCA) protein assay kit, Immunol Fluorescence Staining Kit, and Immunohistochemistry Staining Kit were obtained from Beyotime (Shanghai, China); anti-SQSTM1/P62, anti-BECN, anti-LC3 II, anti-beta amyloid, anti-BACE1, anti-synapsin1, anti-Parkin, anti-Pink1, anti-APPL, and anti-PS1 were obtained from Abcam (Cambridge, UK).
3
Evaluation of Autophagy and Apoptosis Signaling
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The renal cortex tissue and podocytes were lysed with RIPA Lysis Buffer, which then was centrifuged at 12000r/min for 15min at 4°C. The protein concentration was detected using BCA method. The proteins were separated through electrophoresis and transferred into nitrocellulose membrane. The primary antibodies (anti-ATG12, Anti-ATG5, anti-mTOR, anti-Bcl-2, anti-Bax, anti-Caspase-3, anti-GADPH, 1:1000, Abcam, England) (anti-Beclin-1, anti-LC3II: 1:1000. Santa Cruz, America) (Abcam, America) (Cleaved-caspase3: 1:500, Abcam, America) were incubated with the membrane at 4°C overnight blocked by 5% skim milk. The secondary antibody (1:5000) was then incubated with membrane for 2h. The gray value of protein was analyzed using Image Lab 3.0 software and the relative expression was calculated in the ratio of the target protein and reference.
4
Protein Expression Analysis via Western Blot
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Whole cell lysates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). Western blotting was performed using anti-LC3I (1:1000, Abcam, USA), anti-LC3II (1:1000, Abcam, USA), anti-PI3K (1:1000, CST), anti-phospho-PI3K (1:500, CST), anti-Akt (1:3000, CST), anti-phospho-Akt (1:1000, CST), anti-mTOR (1:3000, CST), anti-phospho-mTOR (1:1000, CST), anti-phospho-p50 NF-ĸB (1:1000, CST), p50 NF-ĸB (1:2000, CST), anti-phospho-p65 NF-ĸB (1:1000, CST), and anti-p65 NF-ĸB (1:2000, CST) antibodies. GAPDH (1:10000, Abcam, USA) was used as a loading control for proteins. The band intensities were analyzed using an ECL Plus detection system (Thermo Scientific, Pittsburgh, PA, USA).
5
Exosome Modulation of Trophoblast Autophagy
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Trophoblasts were cultured with or without purified AD-MSC-derived exosomes for 24 h (10 μg/ml). Then, all the cells were gathered and separated, and later for 60 min, they were fixed with 4% paraformaldehyde. The fixed cells were cut into 4-μm sections and embedded in paraffin. Later, they were washed in PBS for three times and blocked with 10% goat serum for 1 h. After that in 0.2% Triton X-100 the sections were washed twice. Next, they were incubated with primary antibodies (anti-LC3-II and anti-tubulin were purchased from Abcam, Cambridge, MA, United States), secondary antibodies (Invitrogen) and DAPI (Guangzhou RiboBio, Guangzhou, China). Using a fluorescence microscope, images were captured.
6
Neural Stem Cell Culture Protocol
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NSCs culture medium was under support from Chi Scientific (Jiangsu, China). Anti-NGF, anti-LC3-II, anti-Beclin1, anti-P62, anti-NeuN, donkey anti-rabbit IgG and anti-mouse IgG with Alexa Fluor 594 (red) and Alexa-Fluor 488 (green) were supplied by Abcam (Cambridge, Britain). DAPI (4′,6-diamidino-2-phenylindole), a fluorescent agent used for nuclear staining, was purchased from Sigma-Aldrich (St. Louis, MO, USA).
7
Western Blot Analysis of Autophagy and Fibrosis Markers
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RIPA lysis buffer was used to extract total protein from mesangial cells and homogenates of the cortical tissues. Protein concentration was determined using a BCA protein assay (Thermo Fisher Scientific). Equal amounts of protein (40 μg/lane) were separated by 10% SDS-PAGE gels and transferred to PVDF membrane (Millipore, USA). Membranes were blocked in 5% non-fat milk for 1 h. Membranes were then incubated with the following antibodies at 4 °C overnight: anti-LC3I and anti-LC3II (1:1000, Abcam), anti-Beclin1 (1:1000, Abcam), anti-p62 (1:1,000, Abcam), anti-TGF beta-1 (1:1000, Abcam), anti-fibronectin (1:1000, Abcam), anti-ASK1 (1:1000, Abcam), anti-collagen IV (1:1000, Abcam), anti-α-SMA (1:400, Abcam), anti-E-cadherin (1:1000, Abcam), anti-N-cadherin (1:1000, Abcam), anti-p-Akt (1:1000, Abcam), anti-p-mTOR (1:1000, Abcam), anti-Akt (1:1000, Abcam), anti-mTOR (1:1000, Abcam), and anti-GAPDH (1:5000, Abcam, Cambridge, UK). Membranes were washed with TBST (Beyotime Biotechnology, Shanghai, China) and incubated with horseradish peroxidase (HRP)-labelled secondary antibodies (1:20,000, BOSTER, Beijing, China) at room temperature for 45 min. Target signals were observed with enhanced chemiluminescence (ECL) detection (Pharmacia; Piscataway-USA).
8
Western Blot Analysis of Neuroblastoma Cells
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SK-N-AS, BE(2 (link))C, SK-N-DZ, SK-N-F1 and SHEP1 cells were digested with trypsin and washed twice with PBS. Cells were harvested and suspended in 1% radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein concentrations were measured using a Bicinchoninic Acid protein assay kit (Beyotime Institute of Biotechnology). Total protein (50 µg) was separated using SDS-PAGE (30% gel) and then the proteins were transferred onto polyvinylidene difluoride membranes. Following blocking for 2 h with 5% goat serum (diluted with PBS) at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-TP53BP2 (1:1,000, catalog no. ab181377; Abcam), anti-LC3 II (1:1,000; catalog no. ab48394; Abcam) and anti-tubulin (1:5,000; catalog no. ab7291; Abcam). The membranes were than incubated at room temperature for 2 h with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:10,000; catalog no. 04-18-06) or horseradish peroxidase-conjugated rabbit anti-goat IgG secondary antibody (1:10,000; catalog no. 14-13-06; both from KPL, Inc., Gaithersburg, MD, USA). Proteins were visualized using BeyoECL Plus reagent (Beyotime Institute of Biotechnology). Western blot data were quantified with Gel-Pro Analyzer 4 software (Media Cybernetics, Inc., Rockville, MD, USA).
9
Immunofluorescence Staining for LC3-II
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The cells were washed with ice-cold PBS, and 4% paraformaldehyde was fixed for 15 min at room temperature. Next, the cells were permeabilized with 0.2% Triton X-100. The cells were incubated with anti-LC3-II (1 : 100, Abcam) for 2 h at room temperature. The cells were washed three times with cold PBS and incubated with an anti-rabbit secondary antibody (1 : 800) (Invitrogen, USA) for 1 h at room temperature. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) and observed by fluorescence microscopy (OLYMPUS-IX73P2F).
10
Immunofluorescent Visualization of Autophagy Marker LC3II
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The cells were fixed with 100% methanol for 5 min at room temperature and then permeabilized with Triton X-100 (3 g/L) for 30 min. The primary antibody (anti-LC3II; Abcam) was used with cells overnight at 4°C. Next, the secondary antibody (an Alexa Fluor ® 488 goat antirabbit secondary antibody; Abcam) was added for incubation for 30 min at room temperature. DAPI was used to stain the nucleus. Anti-fluorescent quenching agent was added to mount the sections. The cells were observed under a fluorescence microscope (Olympus Corp.).
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