Legumain/Asparaginyl Endopeptidase Antibody [Unconjugated]

Western blot shows lysates of J774A.1 mouse reticulum cell sarcoma macrophage cell line and Neuro-2A mouse neuroblastoma cell line. PVDF membrane was probed with 0.2 µg/mL of Sheep Anti-Mouse Legumain/Asparaginyl Endopeptidase Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2058) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). Specific bands were detected for Legumain/Asparaginyl Endopeptidase at approximately 37 and 56 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

The presence of VDAC1-DC in ccRCC cells decreases or abolishes ciliation. A, Triple immunofluorescence labeling and merged images with acetylated alpha -tubulin (Acet. alpha -tubulin in red), Arl13b (in green) and DAPI (in blue). B, Electron microscopy of RCC4+pVHL cells. C, Quantitative analysis of the ciliation percentage was assessed by confocal fluorescence microscopy (n=100-300 cells). D, Both cell lines were seeded at the same density and incubated in Nx for 48h with or without serum. Percentage of ciliated cells, proliferation and FACS analysis were measured. The mean ± SEM is representative of three independent experiments carried out in duplicate. E, F and G, RCC4 cells were transfected with control siRNA (siCtl), (E) siHIF-1 alpha , siHIF-2 alpha and siHIF-1/2 alpha , (F) siVDAC1 and (G) siLGMN. Cell lysates were analyzed by immunoblotting for HIF-1 alpha , HIF-2 alpha , VDAC1, LGMN and beta - tubulin/Actin or HSP90 were used as a loading control. Quantitative analysis of the ciliation percentage was assessed by confocal fluorescence microscopy (n=100-300 cells). A * p<0.05 shows significant differences. Quantification of VDAC1 and VDAC1-delta C protein levels (E). Experiments have been proceeded without serum. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/32194829), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Mu
• Applications: WB
• Clonality: Polyclonal
• Host: Sheep
• Conjugate: Unconjugated
• Concentration: LYOPH

Legumain/Asparaginyl Endopeptidase Antibody [Unconjugated] Summary

• Immunogen: Mouse myeloma cell line NS0-derived recombinant mouse Legumain
  Val18-Tyr435
  Accession # O89017
• Specificity: Detects mouse Legumain in direct ELISAs and Western blots.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Sheep
• Gene: LGMN
• Purity Statement: Antigen Affinity-purified

Applications/Dilutions

• Dilutions:
  • Western Blot 0.2 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Legumain/Asparaginyl Endopeptidase Antibody [Unconjugated]

• AEP
• Asparaginyl Endopeptidase
• cysteine protease 1
• Legumain
• LGMN
• LGMN1
• Protease, cysteine 1
• protease, cysteine, 1 (legumain)
• PRSC1EC 3.4.22.34

Background

Legumain is a lysosomal cysteine protease found in all mouse tissues examined, but was particularly abundant in kidney and placenta (1). Legumain plays a pivotal role in the endosomal/lysosomal degradation system because the Legumain deficiency causes the accumulation of pro cathepsins B, H and L, another group of lysosomal cysteine proteases (2). Over-expression of Legumain in tumors is significant for invasion/metastasis (3). Also known as asparaginyl endopeptidase, it specifically cleaves peptide bonds with Asn at the P1 position. Nevertheless, it also cleaves peptide bonds with Asp at the P1 position. Auto-activation of pro Legumain involves both types of cleavage, which results in the removal of the pro peptides in both C- and N-termini (4). In addition, Legumain activates pro MMP-2 and processes bacterial antigens for MHC class II presentation and pro thymosin alpha to thymosin alpha ₁ and thymosin alpha ₁₁, two acidic peptides with immunoregulatory properties (5-7).

1. Chen, J.M. et al. (1998) Biochem. J. 335:111.
2. Shirahama-Noda, K. et al. (2003) J. Biol. Chem. 278:33194.
3. Liu, C. et al. (2003) Cancer Res. 63: 2957.
4. Li D.N. et al. (2003) J. Biol. Chem. 278:38980.
5. Chen, J.M. et al. (2001) Biol. Chem. 382:777.
6. Schwarz, G. et al. (2002) Biol. Chem. 383:1813.
7. Sarndeses, C.S. et al. (2003) J. Biol. Chem. 278:13286.

Limitations

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

Publications for Legumain/Asparaginyl Endopeptidase Antibody (AF2058)(13)

Publications using AF2058

• Cason SE, Mogre SS, Holzbaur ELF, Koslover EF. Spatiotemporal analysis of axonal autophagosome-lysosome dynamics reveals limited fusion events and slow maturation Molecular Biology of the Cell 2022-11-01 [PMID: 36044338]
• L Cao, C Xu, P Yi, H Li, Y Lin, G Cai, S Wang, D Ou, M Li, J Chen Asparaginyl endopeptidase (AEP) regulates myocardial apoptosis in response to radiation exposure via alterations in NRF2 activation American journal of cancer research, 2021-04-15;11(4):1206-1225. 2021-04-15 [PMID: 33948354] (IHC, Western Blot, Mouse)
• C Chen, D Wang, Y Yu, T Zhao, N Min, Y Wu, L Kang, Y Zhao, L Du, M Zhang, J Gong, Z Zhang, Y Zhang, X Mi, S Yue, X Tan Legumain promotes tubular ferroptosis by facilitating chaperone-mediated autophagy of GPX4 in AKI Cell Death & Disease, 2021-01-11;12(1):65. 2021-01-11 [PMID: 33431801] (Western Blot, Mouse)
• L Fabbri, M Dufies, S Lacas-Gerv, B Gardie, S Gad-Lapite, J Parola, N Nottet, M Meyenberg, J Contenti, D Borchielli, JM Ferrero, NR Leclercq, D Ambrosetti, B Mograbi, S Richard, J Viotti, E Chamorey, N Sadaghianl, M Rouleau, WJ Craigen, B Mari, S Clavel, G Pagès, J Pouysségur, F Bost, NM Mazure Identification of a new aggressive axis driven by ciliogenesis and absence of VDAC1-deltaC in clear cell Renal Cell Carcinoma patients Theranostics, 2020-02-03;10(6):2696-2713. 2020-02-03 [PMID: 32194829] (Western Blot, Mouse)
• Shuke Nie, Yang Tan, Zhentao Zhang, Guiqin Chen, Jing Xiong, Dan Hu et al. Bilateral Implantation of Shear Stress Modifier in ApoE Knockout Mouse Induces Cognitive Impairment and Tau Abnormalities Frontiers in Aging Neuroscience 2018-10-04 [PMID: 30337867]
• Wen Hu, Yunn Chyn Tung, Yanchong Zhang, Fei Liu, Khalid Iqbal Involvement of Activation of Asparaginyl Endopeptidase in Tau Hyperphosphorylation in Repetitive Mild Traumatic Brain Injury Journal of Alzheimer's Disease 2018-07-03 [PMID: 29889065]
• T Yamane, M Kozuka, Y Yamamoto, Y Nakano, T Nakagaki, I Ohkubo, H Ariga Protease activity of legumain is inhibited by an increase of cystatin E/M in the DJ-1-knockout mouse spleen, cerebrum and heart Biochem Biophys Rep, 2017-01-05;9(0):187-192. 2017-01-05 [PMID: 28956004] (Western Blot, Mouse)
• Yang Tan 7,8-Dihydroxyflavone Ameliorates Cognitive Impairment by Inhibiting Expression of Tau Pathology in ApoE-Knockout Mice Front Aging Neurosci, 2016-11-29;8(0):287. 2016-11-29 [PMID: 27965573] (Western Blot, Mouse)
• Lifei Hou, Jessica Cooley, Richard Swanson, Poh Chee Ong, Robert N. Pike, Matthew Bogyo et al. The protease cathepsin L regulates Th17 cell differentiation Journal of Autoimmunity 2015-12-01 [PMID: 26343333] (Western Blot, Mouse)
• L Gangoda, M Doerflinger, R Srivastava, N Narayan, L E Edgington, J Orian et al. Loss of Prkar1a leads to Bcl-2 family protein induction and cachexia in mice Cell Death & Differentiation 2014-11-01 [PMID: 25012505]
• Laura E. Edgington, Martijn Verdoes, Alberto Ortega, Nimali P. Withana, Jiyoun Lee, Salahuddin Syed et al. Functional Imaging of Legumain in Cancer Using a New Quenched Activity-Based Probe Journal of the American Chemical Society 2013-01-09 [PMID: 23215039]
• Zeeuwen PL, van Vlijmen-Willems IM, Cheng T The cystatin M/E-cathepsin L balance is essential for tissue homeostasis in epidermis, hair follicles, and cornea. FASEB J., 2010-05-21;24(10):3744-55. 2010-05-21 [PMID: 20495178] (Western Blot, Mouse)
• Clerin V, Shih HH, Deng N, Hebert G, Resmini C, Shields KM, Feldman JL, Winkler A, Albert L, Maganti V, Wong A, Paulsen JE, Keith JC, Vlasuk GP, Pittman DD Expression of the cysteine protease legumain in vascular lesions and functional implications in atherogenesis. Atherosclerosis, 2008-02-21;0(0):. 2008-02-21 [PMID: 18377911] (ICC, IHC-P, Mouse)

Bioinformatics

• Gene Symbol: LGMN
• Uniprot:
  • O89017()