F12 (Coon’s modification) medium and BFA (brefeldin A) were purchased from Sigma–Aldrich. BFA was stored at −20°C as a 10 mg/ml stock in DMSO. Lactacystin was stored as a 5 mM stock at −20°C. MG-132 (Cell Signaling Technology) was stored at −20°C as a 10 mM stock in DMSO. FBS and newborn calf serum were purchased from Gemini Bio-Products. TaqSelect DNA polymerase was purchased from Lucigen. High-speed plasmid mini kits were from MidSci. HRP (horseradish peroxidase)-conjugated secondary antibodies and Western Lightning ECL reagent were purchased from Sigma–Aldrich and PerkinElmer respectively. Alexa Fluor®-conjugated secondary antibodies were purchased from Invitrogen. Monoclonal antibodies against the V5 epitope tag were from AbD Serotec or against mannosidase II were purchased from Covance. ProteoStat® was purchased from Enzo Life Sciences and cells were stained according to the manufacturer’s instructions. LC3A/B antibodies were from Cell Signaling Technology. Anti-MAL2 polyclonal antibodies were generated and affinity-purified as described previously [2 (link)]. Polyclonal antibodies against haptoglobin, HA321, CE9, rat serum albumin, APN (aminopeptidase N) and lysosomal glycoprotein 120 were provided by Dr A. Hubbard (Johns Hopkins University School of Medicine, Baltimore, MD, U.S.A.).
Lc3a b antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The LC3A/B antibody is a primary antibody that recognizes the microtubule-associated protein 1A/1B-light chain 3 (LC3A and LC3B) proteins. LC3 proteins are involved in the formation of autophagosomes, which are key components of the autophagy process. The LC3A/B antibody can be used to detect and quantify the levels of these proteins in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Aperio scanscope, by Leica (2 mentions)
Tecnai g2 spirit transmission electron microscope, by Thermo Fisher Scientific (2 mentions)
Mhc 1, by Developmental Studies Hybridoma Bank (2 mentions)
Mhc iia, by Developmental Studies Hybridoma Bank (2 mentions)
Mhc2x, by Developmental Studies Hybridoma Bank (2 mentions)
Mhc2b, by Developmental Studies Hybridoma Bank (2 mentions)
Cox4 i1 antibody, by R&D Systems (2 mentions)
β actin antibody, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail, by Roche (2 mentions)
Brefeldin a bfa, by Merck Group (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Proteostat, by Enzo Life Sciences (1 mentions)
Newborn calf serum, by Gemini Bio (1 mentions)
Mg132, by Cell Signaling Technology (1 mentions)
Zen black edition software, by Zeiss (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit secondary antibody, by Abcam (1 mentions)
Nuclear dye dapi, by Solarbio (1 mentions)
6e10 antibody, by BioLegend (1 mentions)
22 protocols using lc3a b antibody
1
Antibody-based detection and analysis
2
Visualizing Mitochondria and Autophagosomes
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Cells seeded on 8-well microscopy
slides were fixed with 2% glutaraldehyde solution (cat. no. 340855,
Sigma Aldrich) by incubating at 4 °C for 15 min. 0.1 M glycine
solution was applied to quench residual aldehydes for 1 h at room
temperature that was followed by permeabilization with 0.25% Triton
X-100 in PBS at room temperature for 15 min. Prior to antibody incubation,
cells were blocked with 3% Blocker BSA in PBS (cat. no. 37525, Thermo
Fisher Scientific) by incubating for 1 h at room temperature. Mitochondria
were labeled by incubating the cells at 4 °C overnight with an
Alexa Fluor 488-conjugated anti-mitochondria antibodies (cat. no.
MAB1273A4, Merck Millipore) in blocking solution at 1:500 dilution.
For imaging autophagosome formation, slides were incubated overnight
with LC3A/B antibodies (Cell Signaling Technologies, polyclonal) at
1:200 dilution followed by incubating with an Alexa Fluor 488-conjugated
donkey anti-rabbit secondary antibody (Abcam, polyclonal) for an hour
at room temperature. Slides were mounted with Antifading Mounting
Medium with DAPI (cat. no. SCR-038448, Dianova) and visualized under
a Zeiss LSM 880 Airyscan inverted microscope at 63× magnification
with immersion oil. Images were obtained using Zeiss Zen software
(Black Edition).
slides were fixed with 2% glutaraldehyde solution (cat. no. 340855,
Sigma Aldrich) by incubating at 4 °C for 15 min. 0.1 M glycine
solution was applied to quench residual aldehydes for 1 h at room
temperature that was followed by permeabilization with 0.25% Triton
X-100 in PBS at room temperature for 15 min. Prior to antibody incubation,
cells were blocked with 3% Blocker BSA in PBS (cat. no. 37525, Thermo
Fisher Scientific) by incubating for 1 h at room temperature. Mitochondria
were labeled by incubating the cells at 4 °C overnight with an
Alexa Fluor 488-conjugated anti-mitochondria antibodies (cat. no.
MAB1273A4, Merck Millipore) in blocking solution at 1:500 dilution.
For imaging autophagosome formation, slides were incubated overnight
with LC3A/B antibodies (Cell Signaling Technologies, polyclonal) at
1:200 dilution followed by incubating with an Alexa Fluor 488-conjugated
donkey anti-rabbit secondary antibody (Abcam, polyclonal) for an hour
at room temperature. Slides were mounted with Antifading Mounting
Medium with DAPI (cat. no. SCR-038448, Dianova) and visualized under
a Zeiss LSM 880 Airyscan inverted microscope at 63× magnification
with immersion oil. Images were obtained using Zeiss Zen software
(Black Edition).
3
Immunofluorescence Staining of LC3A/B
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Cells were collected by cytospin on glass slides and fixed with 4% paraformaldehyde for 15 min. After washing in 10% PBS, the slides were permeabilized with 0.2% Triton X-100 in PBS and then were blocked with 4% bovine serum albumin (#A8020; Solarbio, Shanghai, China) for 1 h at room temperature. After incubated with LC3A/B antibody (#12741, 1:1000, Cell Signaling Technology, Danvers, USA) overnight at 4 °C, the slides were washed with PBS and then incubated with Alexa Fluor 647s antibody (#bs-0295G-AF647, Bioss, Woburn, USA). The cells were stained with nuclear dye DAPI (#C0080; Solarbio, Shanghai, China). All the images were captured with the fluorescence microscope, and representative images were shown.
4
Comprehensive Skeletal Muscle Analysis
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H&E, oil red O, SDH, and myosin heavy chain (MHC) staining were performed as previously described (Ahn et al., 2016 (link); Gan et al., 2013 (link); Jaspers et al., 2014 (link); Zechner et al., 2010 (link)). The fiber typing was done using the antibodies MHC-I (red, BA-D5), MHC-IIA (yellow, 2F7), MHC2x (black, unstained), and MHC2b (green, 10F5) from Developmental Studies Hybridoma Bank (East Iowa City, IA) (Wende et al., 2007 (link)). Autophagy marker LC3A/B antibody (Cell Signaling Technology, 4108) and mitochondrial marker COX4-I1 antibody (R&D Systems, AF5814) were used for co-localization staining in muscle sections. The images were captured using Aperio Scanscope (Leica). For transmission electron microscopy, skeletal muscles were fixed with 1% glutaraldehyde solution. The muscle samples were cut and processed as described in Bal et al. (2016) (link) and Zechner et al. (2010) (link). Electron micrographs were obtained using a Tecnai G2 Spirit transmission electron microscope (FEI, Hillsboro, OR). ImageJ (NIH) was used to quantify images.
5
Targeted Detection of APP and Tau Markers
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Chemicals, including ARV-825 and JQ1, were from Medchemexpress (Catalog #s: HY-16954 and HY-13030, respectively) and their stocks were in DMSO. The G12A antibody was used to detect APP full length and C-terminal fragments (APP-CTFs) (rabbit polyclonal, clone C7 targeting amino acid residues 732–751 of APP751, custom made by Thermo Fisher Scientific) and were previously reported (57 (link), 58 (link), 59 (link)). The sAPPβ antibody was purchased from IBL (Catalog #: 18975). The 6E10 antibody (BioLegend) is a monoclonal antibody (mAb) reactive to amino acid residues 1–16 of Aβ from the N-terminal sequence and can detect sAPPα in medium. The nicastrin (NCT) antibody (Catalog #: 5665), BACE1 antibody (Catalog #: 5606), and LC3 a/b antibody (Catalog #: 12741) were purchased from Cell Signaling. The BRD4 antibody was from BETHYL (Catalog #: A700-004-T). The β-actin antibody was from Sigma and used as a protein control. The antibody for pTau (S396) was from Cell Signaling (Catalog #: 9632). The HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit) were from Pierce. The dilution factors for primary and secondary antibodies were 1: 1000 and 1: 10,000, respectively.
6
Cell Lysis and Western Blotting Protocol
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Cells were lysed in TEGN buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 420 mM NaCl, 10% glycerol, and 0.5% Nonidet P-40) containing proteases inhibitor cocktail (Roche) and 1 mM dithiothreitol (DTT). For Western blotting, the cell lysates were boiled in protein sample buffer (2 M β-mercaptoethanol, 12% sodium dodecyl sulfate (SDS), 0.5 M Tris, pH 6.8, 0.5 mg/mL bromophenol blue, and 30% glycerol), and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). Antibodies used were the following: LC3A/B antibody (#4108; Cell Signaling, Beverly, MA, USA), actin (A2066; Sigma), cleaved caspase-3 (#9661; Cell Signaling), PARP (#9542; Cell Signaling).
7
Autophagy Regulatory Protein Expression
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LC3A/B antibody (Cell Signaling Technology, Beverly, Massachusetts, USA), anti-p62 antibody (Sigma-Aldrich St. Louis, Missouri, USA), Beclin-1 antibody (Abcam Trading (Shanghai) Company, China), Bcl-2 antibody (Abcam Trading (Shanghai) Company, China), Caspase-3/Cleaved-Caspase-3 antibody (Cell Signaling Technology, Beverly, Massachusetts, USA), β-Actin antibody (Cell Signaling Technology, Beverly, Massachusetts, USA), IgG Fc horseradish peroxidase (HRP) secondary antibody (Abcam Trading (Shanghai) Company, China).
8
Immunofluorescence Analysis of Autophagy and Transcription Factors
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Cells were seeded at a density of 5 × 105 cells per well in six well plates containing cover glasses/slips in each well. Following indicated transfections, serum starvation and time points, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) and then permeabilized in pre-chilled 100% methanol for 10 minutes at −20 °C. Cells were then washed with 1x PBS for 5 minutes, blocked and incubated overnight at 4 °C with antibodies raised against LC3A/B antibody (Cell Signaling Technology), FLAG tag (Cell Signaling Technology), NRF2 (Novus Biologicals) and Histone H2B (Abcam). Conjugated secondary antibodies used include goat anti-mouse and anti-rabbit IgG (H + L) Fluorescein (FITC) (Jackson ImmunoResearch Labs) as well as anti-rabbit and anti-mouse IgG Alexa Fluor® 555 conjugates (Cell Signaling Technology). Fixed cells were incubated with diluted secondary antibodies for 1 hour at room temperature after which they were washed three times with 1x PBS and mounted with ProLong Gold Antifade Reagent (Cell Signaling Technology). Analysis was carried out in an epifluorescence microscope using single interference filters set for green (fluorescein isothiocyanate, FITC), red (Texas Red), and blue (DAPI) as well as dual (red/green) band pass filters.
9
Betulinic Acid: Apoptosis and mTOR Pathway Modulation
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Betulinic acid was purchased from YuanYe biotechnology (Shanghai, USA) and dissolved in DMSO as 100 mM. Counting Kit-8 (CCK-8) assay and Annexin V-FITC Apoptosis kit were obtained from BestBio Company (Shanghai, China). mTOR antibody (ab2732), Caspase-3 antibody (ab2302), p62 antibody (ab155686) were provided by Abcam. After that, S6K1 antibody (CST 9202), p-S6K1 antibody (CST 9204S), AMPK antibody (CST 2532S), p-AMPKα1 antibody (CST 2537), p-mTOR antibody (CST 5536S), Caspase8 antibody (CST 4790), Bax antibody (CST 5023S) and LC3A/B antibody (CST 12741) were bought from Cell Signaling Technology. Bcl2 antibody (12789–1-AP) and GAPDH antibody (AP0063) were acquired from Proteintech and Bioworld Technology, respectively.
10
Immunofluorescence Analysis of Cardiomyocytes
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For immunofluorescence analysis of NRCMs, cultured NRCMs were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X‐100 in PBS for 10 minutes, blocked with 3% BSA solution for 1 hour and incubated with an anti‐actin (α‐sarcomeric) antibody (Sigma‐Aldrich; 1:200) or a microtubule‐associated protein 1 light chain 3 (LC3) A/B antibody (Cell Signaling Technology; 1:200) overnight at 4°C. Then, the cells were washed and stained with fluorescence‐conjugated secondary antibodies (Beyotime Biotechnology; Alexa Fluor 647 or Alexa Fluor 488, respectively). Immunofluorescence was analysed with a Nikon A1 confocal microscope (Nikon Corporation), and the cell surface areas and numbers of LC3 puncta were measured using Image‐Pro Plus 6.0 software.
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