LC3 Antibody Pack

Knockout Validated: LC3 Antibody Pack [NB910-40435] - Lysates of HeLa parental cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM Chloroquine for 18 hours. PVDF (Polyvinylidene difluoride) membrane was probed with 0.5 ug/mL of Rabbit Anti-LC3B Polyclonal Antibody (Catalog # NB100-2220) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog# HAF008). A specific band was detected for LC3B at a molecular weight of approximately 15 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. GAPDH is shown as a loading control. This experiment was conducted under reducing conditions. LC3B Antibody [NB100-2220]

Immunohistochemistry: LC3 Antibody Pack [NB910-40435] - Rapamycin increases autophagy in brains of PDAPP mice. Representative epifluorescent (c200x) image of hippocampal CA1 in control- and rapamycin-fed transgenic PDAPP mice stained with an anti-LC3 antibody. An increase in LC3-immunoreactive puncta was observed in CA1 projections of transgenic PDAPP mice following rapamycin administration. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0009979) licensed under a CC-BY license. LC3A Antibody [NB100-2331]

Knockout Validated: LC3 Antibody Pack [NB910-40435] - LC3B was detected in immersion fixed Cloroquine treated HeLa cells (left) but was not detected in LC3B knockout HeLa cells (right) using rabbit anti-human LC3B polyclonal antibody (Catalog #NB100-2220) at 0.3 ug/mL for 3 hours at room temperature. Cells were stained using the NorthernLights(TM) 557-conjugated anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm.

Western Blot: LC3 Antibody Pack [NB910-40435] - High autophagosome concentration is consumed during early immortalized human mesenchymal stem cell differentiation. Immortalized human mesenchymal stem cells were differentiated under osteogenic conditions (see Materials and methods) and assayed for changes in LC3I and LC3II during a 72-hour window. Cells were differentiated under standard conditions (top) or with addition of 5 uM rapamycin (middle) or 5 nM bafilomycin (bottom) for the first 3 hours of differentiation to modulate autophagy. Immunoblots were performed for LC3 at the indicated time points to assess autophagosome degradation via relative changes in LC3II (lower band; 17 kDa). Studies were repeated three times with similar trends seen consistently. Image collected and cropped by CiteAb from the following publication (//stemcellres.com/content/5/6/140), licensed under a CC-BY license. LC3A Antibody [NB100-2331]

Immunohistochemistry: LC3 Antibody Pack [NB910-40435] - Localization of LC3 by immunohistochemical method. The immunohistochemical staining shows that LC3, CRF, and HIF-1alpha have an overlapping localization in villous and extravillous trophoblast. d: decidua; v: villi; DeVe: decidual vessel. Original magnification 40x. Image collected and cropped by CiteAb from the following publication (//www.hindawi.com/journals/bmri/2013/689768/) licensed under a CC-BY license. LC3B Antibody [NB100-2220]

Immunohistochemistry: LC3 Antibody Pack [NB910-40435] - Brain, Cerebral Cortex, Cell Processes in Gray Matter 40x.

Immunohistochemistry: LC3 Antibody Pack [NB910-40435] - Analysis in PFA fixed NIH/3T3 cells using anti-LC3A antibody. Image from verified customer review. LC3A Antibody [NB100-2331]

• Reactivity: Hu, Mu, Rt, Po, Al, Am, Av, Ba, Bv, Ca, Ch, ChHa, Fi, Gp, Ha, In, Pl, Pm, Pm, Rb, SyHa, Ze
• Applications: WB, Simple Western, ELISA, Flow, Func, ICC/IF, IHC, IP, NULL, PAGE, WB, ChIP, IHC, KD, KO

LC3 Antibody Pack Summary

• Description: This pack contains 1 vial each of: NB100-2220 (0.1 mL) and NB100-2331 (0.1 mL).
• Immunogen: For NB100-2331: A synthetic peptide made to an internal portion of the human LC3 protein sequence (between residues 25-121) [Uniprot: Q9H492].
  For NB100-2220: Polyclonal LC3B Antibody was made to a synthetic peptide made to an N-terminal portion of the human LC3B protein sequence (between residues 1-100) [UniProt# Q9GZQ8].
• Marker: Autophagosome Marker
• Gene: MAP1LC3B

Applications/Dilutions

• Dilutions:
  • Chromatin Immunoprecipitation (ChIP)
  • ELISA
  • Flow Cytometry
  • Immunoblotting
  • Immunocytochemistry/Immunofluorescence
  • Immunohistochemistry Whole-Mount
  • Immunohistochemistry
  • Immunohistochemistry-Frozen
  • Immunohistochemistry-Paraffin
  • Immunoprecipitation
  • Knockdown Validated
  • Knockout Validated
  • Proximity Ligation Assay
  • SDS-Page
  • Simple Western
  • Southern Blot
  • Western Blot
• Application Notes: Antibodies in this pack are validated for the following applications:
  
  NB100-2331: Chromatin Immunoprecipitation, ELISA, Flow Cytometry, Immunoblotting, Immunocytochemistry/Immunofluorescence, Immunohistochemistry, Immunohistochemistry Whole-Mount, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Immunoprecipitation, Simple Western, Southern Blot, Western Blot
  NB100-2220: Chromatin Immunoprecipitation (ChIP), ELISA, Flow Cytometry, Immunoblotting, Immunocytochemistry/Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Immunoprecipitation, Knockdown Validated, Knockout Validated, Proximity Ligation Assay, SDS-Page, Simple Western, Western Blot

Reactivity Notes

Antibodies in this pack are validated for use in the following species:

NB100-2331: Amphibian, Canine, Fish, Human, Mouse, Plant, Rat, Zebrafish
NB100-2220: Alligator, Avian, Bacteria, Bovine, Canine, Chicken, Chinese Hamster, Golden Syrian Hamster, Guinea Pig, Hamster, Human, Invertebrate, Monkey, Mouse, Porcine, Primate, Rabbit, Rat, Zebrafish

Fish reactivity reported in scientific literature (PMID:32818499).

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Alternate Names for LC3 Antibody Pack

• ATG8F
• LC3B
• MAP1A/1BLC3
• map1lc3b
• MAP1LC3B-a
• microtubule associated protein 1 light chain 3 beta

Background

Autophagy (macroautophagy) is a catabolic process which targets intracellular components such as proteins and organelles for degradation. Originally described as a bulk degradation process, current research supports its selective nature (1). Selective autophagy targets specific cellular components for degradation including the endoplasmic reticulum (2) (ER-phagy), mitochondria3 (mitophagy), peroxisomes (3) (pexophagy), ribosomes (4) (ribophagy) and bacteria (5) (xenophagy). Autophagy relies on a newly formed phagophore, a membrane structure which elongates, sequesters cellular content, and fuses to form a double membrane vesicle known as the autophagosome. Fusion of autophagosomes with lysosomes gives rise to the autophagolysosome, where cellular components are degraded by lysosome hydrolases (1).

Autophagic flux is supported by autophagy-related proteins (Atgs) initially identified in yeast (6,7). The core autophagy machinery is comprised of 17 Atg proteins that play specific roles in autophagosome formation. Among these Atg proteins, Atg8 is not only involved in autophagosome formation but also functions in cargo selection. In mammals, several Atg8 homologues have been identified including microtubule-associated protein 1 light chain 3 alpha, beta and gamma - LC3A, LC3B, and LC3C (8) respectively, as well as GABA type A receptor-associated protein (GABARAP), GABARAP-Like1, and GABARAP-Like2 (9). LC3 (predicted molecular weight 14kD) is ubiquitously expressed and undergoes posttranslational processing after synthesis. First, the cysteine protease Atg4 cleaves a carboxy terminal sequence to generate the cytosolic form LC3-I. Next, E1-like (Atg7) and E2-like (Atg3) enzymes conjugate phosphatidylethanolamine to the newly exposed carboxyterminal glycine, generating LC3-II. Finally, the Atg12-Atg5-Atg16L1 complex participates in LC3 lipidation and autophagosome formation (10). LC3B-I to LC3B-II conversion correlates with autophagosome number and is considered the best marker to monitor autophagy.

References

1. Yu, L., Chen, Y., & Tooze, S. A. (2018). Autophagy pathway: Cellular and molecular mechanisms. Autophagy. https://doi.org/10.1080/15548627.2017.1378838

2. Forrester, A., De Leonibus, C., Grumati, P., Fasana, E., Piemontese, M., Staiano, L., ... Settembre, C. (2019). A selective ER-phagy exerts procollagen quality control via a Calnexin-FAM 134B complex. The EMBO Journal. https://doi.org/10.15252/embj.201899847

3. He, X., Zhu, Y., Zhang, Y., Geng, Y., Gong, J., Geng, J., ... Zhong, H. (2019). RNF34 functions in immunity and selective mitophagy by targeting MAVS for autophagic degradation. The EMBO Journal. https://doi.org/10.15252/embj.2018100978

4. Mathai, B., Meijer, A., & Simonsen, A. (2017). Studying Autophagy in Zebrafish. Cells. https://doi.org/10.3390/cells6030021

5. Losier, T. T., Akuma, M., McKee-Muir, O. C., LeBlond, N. D., Suk, Y., Alsaadi, R. M., ... Russell, R. C. (2019). AMPK Promotes Xenophagy through Priming of Autophagic Kinases upon Detection of Bacterial Outer Membrane Vesicles. Cell Reports. https://doi.org/10.1016/j.celrep.2019.01.062

6. Nakatogawa, H., Suzuki, K., Kamada, Y., & Ohsumi, Y. (2009). Dynamics and diversity in autophagy mechanisms: Lessons from yeast. Nature Reviews Molecular Cell Biology. https://doi.org/10.1038/nrm2708

7. Tsukada, M., & Ohsumi, Y. (1993). Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Letters. https://doi.org/10.1016/0014-5793(93)80398-E

8. Wild, P., McEwan, D. G., & Dikic, I. (2014). The LC3 interactome at a glance. Journal of Cell Science. https://doi.org/10.1242/jcs.140426

9. Igloi, G. L. (2001). Cloning, expression patterns, and chromosome localization of three human and two mouse homologues of GABAA receptor-associated protein. Genomics. https://doi.org/10.1006/geno.2001.6555

10. Glick, D., Barth, S., & Macleod, K. F. (2010). Autophagy: Cellular and molecular mechanisms. Journal of Pathology. https://doi.org/10.1002/path.2697

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Antibody Packs are guaranteed for 1 year from date of receipt.

Publications for LC3 Antibody Pack (NB910-40435)(14)

Publications using NB910-40435

• Wang Y, Zhao H, Nie X et al. Zinc application alleviates the adverse renal effects of arsenic stress in a protein quality control way in common carp Environ. Res. 2020-08-17 [PMID: 32818499] (WB, Fish)
• Wang SL, Shao BZ, Zhao SB et al. Intestinal autophagy links psychosocial stress with gut microbiota to promote inflammatory bowel disease Cell Death Dis [PMID: 31564717] (WB, Mouse)
• Ye B, Wang Q, et al. Restoring autophagic flux attenuates cochlear spiral ganglion neuron degeneration by promoting TFEB nuclear translocation via inhibiting MTOR. Autophagy 2019-06-01 [PMID: 30706760] (WB, ICC/IF, Mouse)
• Wu A, Hu P, Lin J et al. Activating Cannabinoid Receptor 2 Protects Against Diabetic Cardiomyopathy Through Autophagy Induction. BMC Cancer. 2018-11-15 [PMID: 30459625] (WB, Mouse)
• Kang I, Lee BC, Lee JY et al. Stem cell-secreted 14,15- epoxyeicosatrienoic acid rescues cholesterol homeostasis and autophagic flux in Niemann-Pick-type C disease. Arch Med Sci. 2018-10-01 [PMID: 30429460] (WB, Mouse)
• Xie Q, Liu M, Yan YF et al. Exogenous tetranectin protects against MPP+-induced neurotoxicity by inhibiting apoptosis and autophagy through p70S6K1 World Neurosurg 2018-10-17 [PMID: 30342268] (WB, Human)
• Xie W, Zheng W, Liu M et al. BRF1 ameliorates LPS-induced inflammation through autophagy crosstalking with MAPK/ERK signaling Genes Dis 2018-09-01 [PMID: 30320187] (ICC/IF, Mouse)
• Song R, Yang Y, Lei H et al. HDAC6 inhibition protects cardiomyocytes against doxorubicin-induced acute damage by improving a-tubulin acetylation J. Mol. Cell. Cardiol. 2018-10-10 [PMID: 30315806] (WB, Rat)
• An Q, Yan W, Zhao Y, Yu K. Enhanced neutrophil autophagy and increased concentrations of IL-6, IL-8, IL-10 and MCP-1 in rheumatoid arthritis Int. Immunopharmacol. 2018-10-09 [PMID: 30312880] (IF/IHC, ICC/IF, Human)
• Bao J, Shi Y, Tao M et al. Pharmacological inhibition of autophagy by 3-MA attenuates hyperuricemic nephropathy Clin. Sci. 2018-10-07 [PMID: 30293967] (WB, Rat)
• Wu Y, Li D, Wang Y, Liu X. Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS. Front. Immunol. 2018-02-13 [PMID: 29487594] (Human)
• Chung H, Choi J, Park S Ghrelin protects adult rat hippocampal neural stem cells from excessive autophagy during oxygen-glucose deprivation Endocr. J. 2017-10-21 [PMID: 29057768] (WB, Rat, Human)
• Zhou Q, Luo D, Li T et al. Bone fracture in a rat femoral fracture model is associated with the activation of autophagy. Exp Ther Med 2015-11-01 [PMID: 26640535] (IF/IHC, Rat)
• Cadwell K, Liu JY, Brown SL et al. A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature;456(7219):259-63. 2008-11-13 [PMID: 18849966]

Reviews for LC3 Antibody Pack (NB910-40435) (3)

Average Rating: 5

(Based on 3 reviews)

We have 3 reviews tested in 1 species: Mouse.

reviewed by: Verified Customer ICC Mouse 02/12/2019

Summary

• Application: Immunocytochemistry
• Sample Tested: Human cell
• Species: Mouse
• Lot: NB100-2341

reviewed by: Liyong Zhang WB Mouse 07/11/2014

Summary

• Application: Western Blot
• Species: Mouse
• Lot: S-2

reviewed by: Sriram Ramanan IHC Mouse 04/29/2014

Summary

• Application: Immunohistochemistry
• Sample Tested: Human Brain
• Species: Mouse
• Lot: NB100-2331

FAQs for LC3 Antibody Pack (NB910-40435). (Showing 1 - 1 of 1 FAQs).

1. Do you have a suggestion regarding the starting point for this antibody's dilution in Western blot?
   • We recommend starting with 2.0ug/ml when using this antibody in Western blot.

Bioinformatics

• Gene Symbol: MAP1LC3B