Anti lc3a b antibody

Manufactured by Cell Signaling Technology

Sourced in United States

The Anti-LC3A/B antibody is a tool for the detection and analysis of the LC3A and LC3B proteins. LC3A and LC3B are involved in the process of autophagy, a cellular mechanism for the degradation and recycling of cellular components. The antibody can be used to identify and quantify the expression of these proteins in various experimental systems.

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LC3A/B (219 citations) Anti-LC3A/B (92 citations) LC3A/B antibody (22 citations) Anti-LC3A (8 citations)

20 protocols using anti lc3a b antibody

Immunoblot and Immunofluorescence Analysis of Cell Death Regulators

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The following primary antibodies were used for immunoblot analysis: anti-β-actin antibody (1:1000 dilution; catalog number 8457; Cell Signaling Technology, Danvers, MA, USA), anti-Histone H3 (1:1000 dilution; catalog number AF0863; Affinity Biosciences, Cincinnati, OH, USA), anti-LC3A/B antibody (1:1000 dilution; catalog number 4108; Cell Signaling Technology), anti-p62 antibody (1:1000 dilution; catalog number 5114; Cell Signaling Technology), anti-RIP3 antibody (1:1000 dilution; catalog number ab56164; Abcam, Cambridge, UK), anti-phosphorylated RIP3 (p-RIP3) antibody (1:1000 dilution; catalog number ab195117; Abcam), anti-TFEB antibody (1:1000 dilution; catalog number A303-673A; Bethyl, Montgomery, TX, USA), anti-Cathepsin B (CTSB) antibody (1:1000 dilution; catalog number ab58802; Abcam), anti-LAMP1 antibody (1:1000 dilution; catalog number ab25630; Abcam), and anti-MLKL antibody (1:1000 dilution; catalog number 37705; Cell Signaling Technology). The following primary antibodies were used for Immunofluorescence staining: anti-TFEB antibody (1:100 dilution; catalog number LS-B5907; LifeSpan BioSciences, Seattle, WA, USA), anti-RIP3 antibody (1:1000 dilution; catalog number ab56164; Abcam), anti-LAMP1 (1:200 dilution; catalog number ab24170; Abcam), and anti-CTSB antibody (1:200 dilution; catalog number ab58802; Abcam).

Li R., Zhao X., Zhang S., Dong W., Zhang L., Chen Y., Li Z., Yang H., Huang Y., Xie Z., Wang W., Li C., Ye Z., Dong Z, & Liang X. (2021). RIP3 impedes transcription factor EB to suppress autophagic degradation in septic acute kidney injury. Cell Death & Disease, 12(6), 593.

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Nanoparticle-Induced Autophagy Visualization

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Cells were seeded on μ-Slides (Ibidi, Martinsried); then, incubated with cell culture media (EMEM 10% FBS) containing different types of nanoparticles either IO-cubes or IO-clusters (both 100 μg/mL) for 24 h at 37 °C and 5% CO₂. Afterwards, cells were fixed in 4% paraformaldehyde in PBS for 10 min, permeabilized in 0.5% TritonX 100 in PBS for 20 min, then labeled with anti-LC3A/B antibody, dilution 1:100 (#12741, Cell Signaling Technology) followed by labeling with anti-rabbit Alexa Fluor 488 conjugated antibody, dilution 1:1000 (#A-11008, Thermo Fisher Scientific). Hoechst 33342 (Thermo Fisher Scientific) was used to label nucleus. Fixed cells were imaged using a spinning disk confocal system IXplore SpinSR (Olympus).

Levada K., Pshenichnikov S., Omelyanchik A., Rodionova V., Nikitin A., Savchenko A., Schetinin I., Zhukov D., Abakumov M., Majouga A., Lunova M., Jirsa M., Smolková B., Uzhytchak M., Dejneka A, & Lunov O. (2020). Progressive lysosomal membrane permeabilization induced by iron oxide nanoparticles drives hepatic cell autophagy and apoptosis. Nano Convergence, 7, 17.

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Western Blot Analysis of LC3 and LAMP1

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Samples were homogenized in ice-cold with radioimmunoprecipitation assay (RIPA) buffer. The homogenates were subsequently centrifuged at 15,000 g for 10 min at 4°C. Then, the samples were subjected to electrophoresis in a 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred on to polyvinylidene fluoride (PVDF) membranes (Millipore, ISEQ00010). Nonspecific binding was blocked with 5% nonfat milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature. Subsequently, the PVDF membranes were incubated at 4°C overnight with rabbit anti-LC3 (1:1000), anti-LC3A/B antibody (12741 Cell Signaling Technology, Danvers, Massachusetts, USA) and mouse anti-LAMP1 antibody (15665 Cell Signaling Technology, Danvers, Massachusetts, USA). Next, the membranes were washed with TBST and incubated with peroxidase-linked goat anti-rabbit IgG (1:5000, BS13278, Bioworld Technology Inc, Minneapolis, Minnesota, USA). Finally, the bound antibodies were visualized by using an the electrogenerated chemiluminescence (ECL) detection system (Vazyme Biotech, E411-04).

Tarique I., Shi Y., Gandahi N.S., Ding B., Yang P., Chen C., Vistro W.A, & Chen Q. (2020). in vivo cellular evidence of autophagic associated spermiophagy within the principal cells during sperm storage in epididymis of the turtle. Aging (Albany NY), 12(10), 8987-8999.

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Immunofluorescence Staining of LC3 and CD68

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LV sections (6 samples/group) were deparaffinized in xylene and rehydrated through gradient ethanol (100% to 70%), permeabilized by 1% Triton X‐100, and immersed in 3% H₂O₂ for 10 minutes to inactivate endogenous peroxidase activity. The sections were blocked with 5% goat serum/1% BSA in PBS for 30 minutes at room temperature and then incubated with anti‐LC3‐A/B antibody (1:400, v/v, dilution, Cell Signaling Technology, Danvers, MA) or anti‐CD68 antibody (1:400, v/v, dilution, Abcam, Cambridge, UK) in 3% goat serum/1% BSA at 4°C overnight. After washing with PBS 3 times, the sections were incubated with FITC‐conjugated (Abcam) secondary antibody (1:1000, v/v, dilution) in 3% goat serum/1% BSA for 30 minutes at room temperature. After being washed with PBS and deionized water, the sections were covered with coverslips by ProlongH Gold antifade reagent with 4,‐diamidino‐2‐phenylindole (DAPI; Life Technologies, Carlsbad, CA).

Wang X., Guo Z., Ding Z, & Mehta J.L. (2018). Inflammation, Autophagy, and Apoptosis After Myocardial Infarction. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 7(9), e008024.

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Murine Colon Epithelial Cell Isolation and Analysis

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Murine colon epithelial cells were isolated according to the protocol by Gracz et al. (66 (link)). A total of 1 × 10⁶ IECs (100 μl) were plated on slides using cytospin. IECs were stained with rabbit monoclonal anti-LC3A/B antibody [1:200; catalog no. 12741, Cell Signaling Technology (CST)] or with rabbit monoclonal anti–E-cadherin antibody (1:200; catalog no. 3195, CST) according to the CST protocol. The remaining cell pellet was homogenized and used for Western blot analysis.

Haq S., Wang H., Grondin J., Banskota S., Marshall J.K., Khan I.I., Chauhan U., Cote F., Kwon Y.H., Philpott D., Brumell J.H., Surette M., Steinberg G.R, & Khan W.I. (2021). Disruption of autophagy by increased 5-HT alters gut microbiota and enhances susceptibility to experimental colitis and Crohn’s disease. Science Advances, 7(45), eabi6442.

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Immunofluorescence Analysis of Autophagy

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All reagents for fixation, washing, and blocking were purchased from Beyotime (Beyotime Biotechnology, China). Pancreatic cancer cells were washed with PBS three times for 5 min each and then fixed for 10 min. They were permeabilized with 0.1% Triton X-100 in PBS for 10 min. They were then blocked for 1 h at room temperature, followed by incubation with anti-LC3A/B antibody (#12741, Cell Signaling, 1:1000) at 4 °C overnight. After washing with PBS extensively the following day, cells were incubated with Cy3-Labeled Goat Anti-Rabbit IgG (Beyotime Biotechnology, China) for 3 h at room temperature. Chromatin was stained with DAPI for 10 min, and cells were observed under a fluorescence microscope (Nikon, Eclipse 80i, Japan).

Yang C., Zhang J.J., Peng Y.P., Zhu Y., Yin L.D., Wei J.S., Gao W.T., Jiang K.R, & Miao Y. (2017). A Yin-Yang 1/miR-30a regulatory circuit modulates autophagy in pancreatic cancer cells. Journal of Translational Medicine, 15, 211.

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Immunofluorescence Microscopy Analysis of LC3 Puncta

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Cells were grown on glass coverslips overnight, fixed with 4% paraformaldehyde (Sigma-Aldrich, P-6148), and treated with 0.2% Triton X-100 to permeabilize for 30 min on ice. To detect LC3 puncta, immunofluorescence microscopy assay was performed with anti-LC3A/B antibody (4108, Cell Signaling Technology) as described previously (24 (link)). Slides were mounted in VECTA SHIELD mounting medium (H-1000, Vector Laboratories, Burlingame, CA, USA) and examined under a LSM 710 laser-scanning confocal microscope (Carl Zeiss, Jena, Germany).

Min Y., Lee S., Kim M.J., Chun E, & Lee K.Y. (2017). Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1. Frontiers in Immunology, 8, 1827.

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Evaluating Cellular Responses to Oxidative Stress

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UM cells MEL270 HEK 293 cells and MCF-7 and RPMI-1640 medium (30–2001) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). Heat-inactivated fetal bovine serum (HI-FBS) (10438–026) and Penicillin-Streptomycin (15140122) were purchased from Life Technologies (Grand Island, NY). Anti-Rock1 antibody (rabbit-4035), anti-Diap1 antibody (rabbit-5486), and anti-p62 (rabbit-5114), anti-YAP1 (rabbit-4912), anti-β-actin antibody (rabbit-2128), anti-LC3A/B antibody was (rabbit-4108) and HRP-linked secondary antibodies- anti rabbit (7074S, 7076S) were obtained from Cell Signaling Technology (Danvers, MA), anti-TEF1 was purchased by Abcam (rabbit-ab133533). Methionine sulfoxide antibody cat #600160 was from Cayman, USA. N-acetyl-L-cysteine and L-histidine were purchased from Sigma-Aldrich (A7250 and H8000 respectively). Verteporfin (Visudyne) was obtained from Novartis (Novartis, Basel, Switzerland) and was dissolved following the manufacture’s protocol.

Konstantinou E.K., Notomi S., Kosmidou C., Brodowska K., Al-Moujahed A., Nicolaou F., Tsoka P., Gragoudas E., Miller J.W., Young L.H, & Vavvas D.G. (2017). Verteporfin-induced formation of protein cross-linked oligomers and high molecular weight complexes is mediated by light and leads to cell toxicity. Scientific Reports, 7, 46581.

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Cryptococcus gattii Infection Assay

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J774 macrophage-like cells were seeded onto 13mm glass coverslips at density of 1 × 10⁵ cells. Macrophages were then incubated at 37°C and 5% CO₂ 24 hours prior to infection. C. gattii strains (CDCF2932, ENV152, CBS8684, CBS7750, CBS1930, CBS7229, CDCR271 and R265) were cultured and J774 cells infected as previously described for the macrophage infection assay. After 2 h of co-incubation, extracellular yeast were removed by extensive PBS washes and, if appropriate, cells were further incubated in assay media at 37°C and 5 % CO₂. Cells were fixed with 4% PFA for 10 min at either 2 h (T=0) or 20 h (T=18) after infection. Coverslips were washed with PBS before treatment with 50 mM NH₄Cl for 10 min and cells were permeabilised in 0.1% Triton X-100 for 4 min. Coverslips were blocked with 5% Goat Serum and 1% BSA in PBS for 1h and incubated with 0.25 μgml⁻¹ rabbit polyclonal anti-LC3A/B antibody (Cell Signaling) for 30 min, followed by 20 min incubation with 3 μg ml⁻¹ goat anti-Rabbit IgG-TRITC (Sigma). Coverslips were then mounted in Mowiol (Calbiochem) with p-phenylenediamine antifade agent. Images were captured using a Nikon eclipse Ti inverted epifluorescence microscope and NIS elements AR 3.2 software (Nikon) with a 60x objective (Plan Apo VC 1.4 NA DIC N2) and camera (Qimaging QICAM-B Mono). Images were analysed using ImageJ software (National Institutes of Health).

Voelz K., Johnston S.A., Smith L.M., Hall R.A., Idnurm A, & May R.C. (2014). ‘Division of labour’ in response to host oxidative burst drives a fatal Cryptococcus gattii outbreak. Nature Communications, 5, 5194.

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Immunofluorescence Assay Reagents

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Reagents were purchased or obtained from the following sources: anti-myosin 1b antibody (ab194356, Abcam); anti-VCAM-1 antibody (sc-13160, Santa Cruz Technology); anti-λH2AX antibody (sc-517348, Santa Cruz Technology); ICAM-1 antibody (sc-8439, Santa Cruz Technology); anti-p21antibody (sc-6246, Santa Cruz Technology); anti-LC3A/B antibody (#4108S, Cell Signaling Technology); anti-LAMP1 antibody (#9091S, Cell Signaling Technology); anti-p62 antibody (18420-1-AP, Proteintech); anti-LRRK2 antibody (#5559S, Cell Signaling Technology); and anti-tubulin antibody (SAB4500087, Sigma-Aldrich). Duolink® In Situ Detection Reagents Red (DUO92008) was from Sigma. IRDye 800-conjugated affinity purified goat anti-rabbit IgG F(c) was purchased from LI-COR Biosciences (Lincoln, Nebraska, USA); goat anti-mouse IgG (H+L) secondary antibody Alexa Fluor® 680 conjugate, goat anti-mouse IgG (H+L) secondary antibody Alexa Fluor® 488 conjugate, goat anti-rabbit IgG (H+L) secondary Antibody Alexa Fluor® 488 conjugate, and goat anti-rabbit IgG (H+L) secondary antibody Alexa Fluor® 594 conjugate were from Invitrogen/Thermo Fisher Scientific (Waltham, MA, USA). Insulin-transferrin-selenite sodium and dexamethasone were from Sigma (St. Louis, Missouri, USA). All cell culture media and materials were purchased from Gibco/Thermo Fisher Scientific (Waltham, Massachusetts, USA).

Yu Y., Ren Y., Li Z., Li Y., Li Y., Zhang Y., Gui R., Cui Y., Qian L, & Xiong Y. (2023). Myo1b Promotes Premature Endothelial Senescence and Dysfunction via Suppressing Autophagy: Implications for Vascular Aging. Oxidative Medicine and Cellular Longevity, 2023, 4654083.

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