LC3 Antibody Pack

Western Blot: LC3 Antibody Pack [NB910-40752] - LC3/MAP1LC3A Antibody Pack [NB910-40752] - Detection of LC3B in treated U87-MG (human glioblastoma astrocytoma) lysates using NB600-1384.

Immunocytochemistry/Immunofluorescence: LC3 Antibody Pack [NB910-40752] - LC3/MAP1LC3A Antibody Pack [NB910-40752] - Immunofluorescent staining of treated U373-MG cells using NB600-1384. The nuclei were stained with DAPI.

Knockout Validated: LC3 Antibody Pack [NB910-40752] - 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 [NB910-40752] - Autophagosomes accumulate in neurons of the VH gray matter at day 1 after SCI. Representative images of IHC staining for LC3 (green) and neuronal marker NeuN (red) in VH of gray matter from sham and SCI animals. Stronger co-localization between NeuN and LC3 is apparent at day 1 after SCI. Scale bar is 20 um. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2014527) licensed under a CC-BY licence. LC3B Antibody [NB100-2220]

Immunohistochemistry: LC3 Antibody Pack [NB910-40752] - Analysis in mouse renal tissue. Image from verifed customer review. LC3A Antibody [NB100-2331]

Western Blot: LC3/MAP1LC3A Antibody Pack [NB910-40752] - Detection of autophagic LC3 in mouse ES cell lysate using NB 100-2331. The atg5-/- lane (ES cells, cultured to form embryonic bodies, that are deficient in conversion of LC3-1 to LC3-11) demonstrates the specificity of NB 100-2331, as there is no detection of LC3-11. Photo courtesy of Dr. Beth Levine, UT Southwestern Medical Center.

Immunohistochemistry: LC3/MAP1LC3A Antibody Pack [NB910-40752] - Staining of treated U373-MG (human glioblastoma) cells using NB600-1384.

• Reactivity: Hu, Mu, Rt, Po, Ba, Bv, Ca, Pm, Ze
• Applications: WB, Simple Western, ChIP, ELISA, Flow, IB, ICC/IF, IHC, IHC-Fr, IHC-P, IP, KD, KO

LC3 Antibody Pack Summary

• Immunogen: For NB600-1384: Polyclonal LC3B Antibody was made to a synthetic peptide made to the N-terminal region of the human LC3B protein [Uniprot: Q9GZQ8].
  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].
• Gene: MAP1LC3B

Applications/Dilutions

• Dilutions:
  • Chromatin Immunoprecipitation
  • ELISA
  • Flow Cytometry
  • Immunoblotting
  • Immunocytochemistry/Immunofluorescence
  • Immunohistochemistry
  • Immunohistochemistry-Frozen
  • Immunohistochemistry-Paraffin
  • Immunoprecipitation
  • Knockdown Validated
  • Knockout Validated
  • Simple Western
  • Western Blot
• Application Notes: Antibodies in this pack are validated for the following applications:
  
  NB600-1384: Electron Microscopy, Flow Cytometry, Immunoblotting, Immunocytochemistry/Immunofluorescence, Immunohistochemistry, Immunohistochemistry Free-Floating, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Immunoprecipitation, Knockdown Validated, Knockout Validated, Simple Western, Western Blot
  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:

NB600-1384: Bacteria, Bovine, Canine, Human, Mouse, Porcine, Primate, Rat, Yeast, Zebrafish
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

Packaging, Storage & Formulations

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

Notes

This pack contains 1 vial each of: NB100-2220 (0.1 mL), NB100-2331 (0.1 mL) and NB600-1384 (0.1 mL).

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 (NB910-40752)(3)

Publications using NB910-40752

• Chao A, Lin CY, Chao AN, Tsai CL et al. Lysine-specific demethylase 1 (LSD1) destabilizes p62 and inhibits autophagy in gynecologic malignancies Oncotarget 2017-09-26 [PMID: 29088798] (WB, ICC/IF, Human)
• Kowalewski B, Heimann P, Ortkras T et al. Ataxia is the major neuropathological finding in arylsulfatase G-deficient mice: similarities and dissimilarities to Sanfilippo disease (mucopolysaccharidosis type III). Hum. Mol. Genet. 2014-12-01 [PMID: 25452429] (WB, Mouse)
  
  Details:
  LC3 antibody used for WB on detergent-soluble fractions of the cerebellum from 12- and 24-month-old arylsulfatase G/Arsg KO mice, a model resembling Sanfilippo syndrome animal models (Figure 6)
• Settembre, C et al. A block of autophagy in lysosomal storage disorders. Hum Mol Genet;17(1):119-29. 2008-01-01 [PMID: 17913701]

FAQs for LC3 Antibody Pack (NB910-40752). (Showing 1 - of FAQs).

Bioinformatics

• Gene Symbol: MAP1LC3B