Recombinant Mouse IL-27 (NS0-expressed) Protein

Carrier Free

Catalog # Availability Size / Price Qty
2799-ML-010/CF

With Carrier

Catalog # Availability Size / Price Qty
2799-ML-010
Best Seller
Recombinant Mouse IL-27 (NS0-expressed) Protein Bioactivity
2 Images
Product Details
Citations (39)
FAQs
Reviews (4)

Recombinant Mouse IL-27 (NS0-expressed) Protein Summary

Product Specifications

Purity
>97%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured in an anti-viral assay using HepG2 human hepatocellular carcinoma cells infected with encephalomyocarditis (EMC) virus. Bender H. et al. (2009) Hepatology 50:585. The ED50 for this effect is 0.6-6 ng/mL.
Source
Mouse myeloma cell line, NS0-derived mouse IL-27 protein
Mouse EBI3
(Tyr19-Pro228)
Accession # O35228
GGGSGGGSGGGSGGGS Mouse p28
(Phe29-Ser234)
Accession # Q8K3I6
6-His tag
N-terminus C-terminus
N-terminal Sequence
Analysis
Tyr19
Predicted Molecular Mass
48.7 kDa
SDS-PAGE
60-65 kDa, reducing conditions

Product Datasheets

You must select a language.

x

2799-ML (with carrier)

You must select a language.

x

2799-ML/CF (carrier free)

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

2799-ML

Formulation Lyophilized from a 0.2 μm filtered solution in PBS and CHAPS with BSA as a carrier protein.
Reconstitution Reconstitute at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin.
Shipping The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.

2799-ML/CF

Formulation Lyophilized from a 0.2 μm filtered solution in PBS and CHAPS.
Reconstitution Reconstitute at 100 μg/mL in PBS.
Shipping The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.

Scientific Data

Bioactivity Recombinant Mouse IL-27 (NS0-expressed) Protein Bioactivity View Larger

Recombinant Mouse IL-27 (Catalog # 2799-ML) demonstrates anti-viral activity in HepG2 human hepatocellular carcinoma cells infected with encephalomyocarditis (EMC) virus. The ED50 for this effect is 0.6-6 ng/mL.

SDS-PAGE Recombinant Mouse IL-27 (NS0-expressed) Protein SDS-PAGE View Larger

1 μg/lane of Recombinant Mouse IL-27 was resolved with SDS-PAGE under reducing (R) conditions and visualized by silver staining, showing a single band at 61 kDa.

Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

=
÷

Background: IL-27

IL-27 is a heterodimeric group 2 receptor ligand molecule that belongs to the IL-6/IL-12 family of long type I cytokines (1). It is composed of EBI3 (EBV-induced gene 3), a 34 kDa glycoprotein that is related to the p40 subunit of IL-12 and IL-23, and p28, the cloned 28 kDa glycoprotein that is related to the p35 chain of IL-12 (2-4). The mouse EBI3 gene encodes a 228 amino acid (aa) precursor that contains an 18 aa signal peptide and a 210 aa mature protein (5). The mature region contains two potential N-linked glycosylation sites, two fibronectin type III domains, and two pairs of conserved cysteine residues that place the molecule in the type I cytokine receptor family (5). As with p40, (the EBI3 counterpart in IL-12), IL-27 EBI3 is reported to form homodimers (6). Mouse EBI3 is 61% and 66% aa identical to human and bovine EBI3. The mouse p28 gene encodes a 234 aa precursor that contains a 28 aa signal sequence and a 206 aa mature region (7). The mature region is characterized by the presence of one potential N-linked glycosylation site and four alpha -helices, placing it in the IL-6 family of helical cytokines. Mouse p28 is 74% aa identical to human p28. IL-27 is expressed by monocytes, endothelial cells and dendritic cells (8). IL-27 binds to and signals through a heterodimeric receptor complex composed of WSX-1 (TCCR) and gp130. Evidence suggests IL-27 interacts only with WSX-1 (7, 9, 10). IL-27 has both anti- and proinflammatory properties. As an anti-inflammatory, IL-27 seems to induce a general negative feedback program that limits T and NK-T cell activity (3, 8). At the onset of infection, IL-27 induces an IL-12 receptor on naïve CD4+ T cells, making them susceptible to subsequent IL-12 activity (and possible Th1 development) (6). Notably, IL-12 family cytokines are both induced and inhibited by bacterial products. Microbes promote IL-27 secretion through TLR4, and also block IL-27 production via C5a induction (11).

References
  1. Boulay, J-L. et al. (2003) Immunity 19:159.
  2. Trinchieri, G. et al. (2003) Immunity 19:641.
  3. Murakami, M. et al. (2004) Growth Factors 22:75.
  4. Cordoba-Rodriguez, R. and D.M. Frucht (2003) Expert Opin. Biol. Ther. 3:715.
  5. Nomura, H. et al. (1997) GenBank Accession # O35228.
  6. Holscher, C. (2004) Med. Microbiol. Immunol. (Berl). 193:1.
  7. Pflanz, S. et al. (2002) Immunity 16:779.
  8. Villarino, A.V. et al. (2004) J. Immunol. 173:715.
  9. Pflanz, S. et al. (2004) J. Immunol. 172: 2225.
  10. Scheller, J. et al. (2005) Biochem. Biophys. Res. Commun. 326:724.
  11. Hawlish, H. et al. (2005) Immunity 22:415.
Long Name
Interleukin 27
Entrez Gene IDs
246778 (Human); 246779 (Mouse); 102121242 (Cynomolgus Monkey)
Alternate Names
IL-27 p28 subunit; IL-27 subunit alpha; IL27; IL-27; IL27A; IL-27-A; IL27-A; IL27p28; IL30; interleukin 27; interleukin 30; interleukin-27 subunit alpha; MGC71873; p28IL-27A

Citations for Recombinant Mouse IL-27 (NS0-expressed) Protein

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

39 Citations: Showing 1 - 10
Filter your results:

Filter by:

  1. Targeting PGLYRP1 promotes antitumor immunity while inhibiting autoimmune neuroinflammation
    Authors: Schnell, A;Huang, L;Regan, BML;Singh, V;Vonficht, D;Bollhagen, A;Wang, M;Hou, Y;Bod, L;Sobel, RA;Chihara, N;Madi, A;Anderson, AC;Regev, A;Kuchroo, VK;
    Nature immunology
    Species: Transgenic Mouse, Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  2. Cell type specific IL-27p28 (IL-30) deletion in mice uncovers an unexpected regulatory function of IL-30 in autoimmune inflammation
    Authors: D Kim, S Kim, MS Kang, Z Yin, B Min
    Scientific Reports, 2023-02-01;13(1):1812.
    Species: Mouse
    Sample Types: In Vivo
    Applications: Bioassay
  3. A critical role for Th17 cell-derived TGF-&beta1 in regulating the stability and pathogenicity of autoimmune Th17 cells
    Authors: G Choi, YJ Park, M Cho, H Moon, D Kim, CY Kang, Y Chung, BS Kim
    Experimental & Molecular Medicine, 2021-05-28;53(5):993-1004.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  4. The Inhibition of Group II Innate Lymphoid Cell Response by IL-27 in Allergic Rhinitis
    Authors: X Luo, Q Zeng, Y Li, Y Tang, W Liu, R Luo
    Journal of Immunology Research, 2020-12-16;2020(0):6661524.
    Species: Mouse
    Sample Types: In Vivo
    Applications: In Vivo
  5. An IL-27-Driven Transcriptional Network Identifies Regulators of IL-10 Expression across T Helper Cell Subsets
    Authors: H Zhang, A Madi, N Yosef, N Chihara, A Awasthi, C Pot, C Lambden, A Srivastava, PR Burkett, J Nyman, E Christian, Y Etminan, A Lee, H Stroh, J Xia, K Karwacz, PI Thakore, N Acharya, A Schnell, C Wang, L Apetoh, O Rozenblatt, AC Anderson, A Regev, VK Kuchroo
    Cell Rep, 2020-11-24;33(8):108433.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Cell Culture
  6. Autocrine TGF-beta1 Maintains the Stability of Foxp3+ Regulatory T Cells via IL-12Rbeta2 Downregulation
    Authors: G Choi, H Na, DS Kuen, BS Kim, Y Chung
    Biomolecules, 2020-05-27;10(6):.
    Species: Transgenic Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  7. Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice
    Authors: M Matsuda, K Doi, T Tsutsumi, M Inaba, J Hamaguchi, T Terada, R Kawata, K Kitatani, T Nabe
    J. Pharmacol. Sci., 2019-11-02;141(4):139-145.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Cell Culture
  8. Activation of aryl hydrocarbon receptor signaling by a novel agonist ameliorates autoimmune encephalomyelitis
    Authors: A Abdullah, M Maged, I Hairul-Isl, A Osama I, H Maha, A Manal, H Hamza
    PLoS ONE, 2019-04-26;14(4):e0215981.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  9. IL-27 targets Foxp3+ Tregs to mediate antiinflammatory functions during experimental allergic airway inflammation
    Authors: QT Nguyen, E Jang, HT Le, S Kim, D Kim, N Dvorina, MA Aronica, WM Baldwin, K Asosingh, S Comhair, B Min
    JCI Insight, 2019-01-24;4(2):.
    Species: Mouse
    Sample Types: In Vivo
    Applications: In Vivo
  10. Indole-3-Pyruvic Acid, an Aryl Hydrocarbon Receptor Activator, Suppresses Experimental Colitis in Mice
    Authors: R Aoki, A Aoki-Yoshi, C Suzuki, Y Takayama
    J. Immunol., 2018-11-14;0(0):.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Differentiation, Differentiation
  11. Notch ligand Delta-like 4 induces epigenetic regulation of Treg cell differentiation and function in viral infection
    Authors: HA Ting, D de Almeida, AJ Rasky, CA Malinczak, IP Maillard, MA Schaller, NW Lukacs
    Mucosal Immunol, 2018-07-23;0(0):.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  12. ??TCR regulates production of interleukin-27 by neutrophils and attenuates inflammatory arthritis
    Authors: L Bouchareyc, EM Grössinger, M Kang, IE Adamopoulo
    Sci Rep, 2018-05-15;8(1):7590.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  13. Treg-specific IL-27R? deletion uncovers a key role for IL-27 in Treg function to control autoimmunity
    Authors: J Do, D Kim, S Kim, A Valentin-T, N Dvorina, E Jang, V Nagarajave, TM DeSilva, X Li, AH Ting, DAA Vignali, SA Stohlman, WM Baldwin, B Min
    Proc. Natl. Acad. Sci. U.S.A., 2017-09-05;0(0):.
    Species: Mouse
    Sample Types: In Vivo
    Applications: In Vivo
  14. The STAT4/MLL1 Epigenetic Axis Regulates the Antimicrobial Functions of Murine Macrophages
    Authors: WF Carson, KA Cavassani, EM Soares, S Hirai, NA Kittan, MA Schaller, MM Scola, A Joshi, A Matsukawa, DM Aronoff, CN Johnson, Y Dou, KA Gallagher, SL Kunkel
    J. Immunol., 2017-07-21;0(0):.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  15. Balanced Bcl-3 expression in murine CD4(+) T cells is required for generation of encephalitogenic Th17 cells
    Authors: IA Mufazalov, J Kuschmann, D Andruszews, J Masri, LA Gabriel, P Adams, S Reissig, N Hövelmeyer, A Waisman
    Eur. J. Immunol., 2017-06-29;0(0):.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  16. STAT1 Represses Cytokine-Producing Group 2 and Group 3 Innate Lymphoid Cells during Viral Infection
    Authors: MT Stier, K Goleniewsk, JY Cephus, DC Newcomb, TP Sherrill, KL Boyd, MH Bloodworth, ML Moore, K Chen, JK Kolls, RS Peebles
    J. Immunol., 2017-06-02;0(0):.
    Species: Mouse
    Sample Types: Cell Lysates
    Applications: Bioassay
  17. Interleukin-27 Early Impacts Leishmania infantum Infection in Mice and Correlates with Active Visceral Disease in Humans
    Authors: Anabela Cordeiro-d
    Front Immunol, 2016-11-04;7(0):478.
    Species: Mouse
    Sample Types: In Vivo
    Applications: In Vivo
  18. IL-27 Directly Enhances Germinal Center B Cell Activity and Potentiates Lupus in Sanroque Mice
    J Immunol, 2016-09-12;0(0):.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  19. Differential Production of Type I IFN Determines the Reciprocal Levels of IL-10 and Proinflammatory Cytokines Produced by C57BL/6 and BALB/c Macrophages
    J Immunol, 2016-08-22;0(0):.
    Species: Mouse
    Sample Types: Cell Culture Supernates
    Applications: Bioassay
  20. Interleukin-27 is elevated in sepsis-induced myocardial dysfunction and mediates inflammation
    Cytokine, 2016-08-12;88(0):1-11.
    Species: Mouse
    Sample Types: In Vivo
    Applications: In Vivo
  21. Thymic DCs derived IL-27 regulates the final maturation of CD4(+) SP thymocytes
    Sci Rep, 2016-07-29;6(0):30448.
    Species: Mouse
    Sample Types: In Vivo, Whole Cells
    Applications: Bioassay, In Vivo
  22. TGF-beta3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.
    Authors: Okamura T, Sumitomo S, Morita K, Iwasaki Y, Inoue M, Nakachi S, Komai T, Shoda H, Miyazaki J, Fujio K, Yamamoto K
    Nat Commun, 2015-02-19;6(0):6329.
    Species: Mouse
    Sample Types: Cell Culture Supernates
    Applications: Bioassay
  23. An IL-27/NFIL3 signalling axis drives Tim-3 and IL-10 expression and T-cell dysfunction.
    Authors: Zhu C, Sakuishi K, Xiao S, Sun Z, Zaghouani S, Gu G, Wang C, Tan D, Wu C, Rangachari M, Pertel T, Jin H, Ahmed R, Anderson A, Kuchroo V
    Nat Commun, 2015-01-23;6(0):6072.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  24. Role of Blimp-1 in programing Th effector cells into IL-10 producers.
    Authors: Neumann C, Heinrich F, Neumann K, Junghans V, Mashreghi M, Ahlers J, Janke M, Rudolph C, Mockel-Tenbrinck N, Kuhl A, Heimesaat M, Esser C, Im S, Radbruch A, Rutz S, Scheffold A
    J Exp Med, 2014-07-29;211(9):1807-19.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  25. IL-27 limits central nervous system viral clearance by promoting IL-10 and enhances demyelination.
    Authors: de Aquino M, Kapil P, Hinton D, Phares T, Puntambekar S, Savarin C, Bergmann C, Stohlman S
    J Immunol, 2014-06-02;193(1):285-94.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  26. miR-20b suppresses Th17 differentiation and the pathogenesis of experimental autoimmune encephalomyelitis by targeting RORgammat and STAT3.
    Authors: Zhu E, Wang X, Zheng B, Wang Q, Hao J, Chen S, Zhao Q, Zhao L, Wu Z, Yin Z
    J Immunol, 2014-05-19;192(12):5599-609.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  27. Timed action of IL-27 protects from immunopathology while preserving defense in influenza.
    Authors: Liu F, Kenngott E, Schroter M, Kuhl A, Jennrich S, Watzlawick R, Hoffmann U, Wolff T, Norley S, Scheffold A, Stumhofer J, Saris C, Schwab J, Hunter C, Debes G, Hamann A
    PLoS Pathog, 2014-05-08;10(5):e1004110.
    Species: Mouse
    Sample Types: In Vivo
    Applications: In Vivo
  28. IL-21 promotes CD8+ CTL activity via the transcription factor T-bet.
    Authors: Sutherland A, Joller N, Michaud M, Liu S, Kuchroo V, Grusby M
    J Immunol, 2013-03-11;190(8):3977-84.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  29. IL-6 activates STAT5 in T cells.
    Authors: Tormo AJ, Letellier MC, Sharma M, Elson G, Crabe S, Gauchat JF
    Cytokine, 2012-07-31;60(0):575.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  30. Regulation of innate CD8+ T-cell activation mediated by cytokines.
    Proc Natl Acad Sci U S A, 2012-06-04;109(25):9971-6.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  31. Interleukin-27 and interferon-gamma are involved in regulation of autoimmune arthritis.
    Authors: Rajaiah R, Puttabyatappa M, Polumuri SK, Moudgil KD
    J. Biol. Chem., 2010-12-01;286(4):2817-25.
    Species: Rat
    Sample Types: In Vivo
    Applications: In Vivo
  32. Interleukin-1 family cytokines as mucosal vaccine adjuvants for induction of protective immunity against influenza virus.
    Authors: Kayamuro H, Yoshioka Y, Abe Y, Arita S, Katayama K, Nomura T, Yoshikawa T, Kubota-Koketsu R, Ikuta K, Okamoto S, Mori Y, Kunisawa J, Kiyono H, Itoh N, Nagano K, Kamada H, Tsutsumi Y, Tsunoda S
    J. Virol., 2010-09-29;84(24):12703-12.
    Species: Mouse
    Sample Types: In Vivo
    Applications: In Vivo
  33. Essential role for IL-27 receptor signaling in prevention of Th1-mediated immunopathology during malaria infection.
    Authors: Findlay EG, Greig R, Stumhofer JS
    J. Immunol., 2010-07-14;185(4):2482-92.
    Species: Mouse
    Sample Types: In Vivo
    Applications: In Vivo
  34. Cutting edge: IL-27 induces the transcription factor c-Maf, cytokine IL-21, and the costimulatory receptor ICOS that coordinately act together to promote differentiation of IL-10-producing Tr1 cells.
    Authors: Pot C, Jin H, Awasthi A
    J. Immunol., 2009-07-01;183(2):797-801.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  35. Expression of WSX1 in tumors sensitizes IL-27 signaling-independent natural killer cell surveillance.
    Authors: Dibra D, Cutrera JJ, Xia X, Birkenbach MP, Li S
    Cancer Res., 2009-06-23;69(13):5505-13.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  36. Amelioration of delayed-type hypersensitivity responses by IL-27 administration.
    Authors: Miyazaki Y, Shimanoe Y, Wang S, Yoshida H
    Biochem. Biophys. Res. Commun., 2008-06-20;373(3):397-402.
    Species: Mouse
    Sample Types: In Vivo
    Applications: In Vivo
  37. The type I IFN induction pathway constrains Th17-mediated autoimmune inflammation in mice.
    Authors: Guo B, Chang EY, Cheng G
    J. Clin. Invest., 2008-05-01;118(5):1680-90.
    Species: Mouse
    Sample Types: In Vivo, Whole Cells
    Applications: Bioassay, In Vivo
  38. Suppression of autoimmune inflammation of the central nervous system by interleukin 10 secreted by interleukin 27-stimulated T cells.
    Authors: Fitzgerald DC, Zhang GX, El-Behi M, Fonseca-Kelly Z, Li H, Yu S, Saris CJ, Gran B, Ciric B, Rostami A
    Nat. Immunol., 2007-11-11;8(12):1372-9.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  39. Suppressive effect of IL-27 on encephalitogenic Th17 cells and the effector phase of experimental autoimmune encephalomyelitis.
    Authors: Fitzgerald DC, Ciric B, Touil T, Harle H, Grammatikopolou J, Das Sarma J, Gran B, Zhang GX, Rostami A
    J. Immunol., 2007-09-01;179(5):3268-75.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay

FAQs

No product specific FAQs exist for this product, however you may

View all Proteins and Enzyme FAQs

Reviews for Recombinant Mouse IL-27 (NS0-expressed) Protein

Average Rating: 5 (Based on 4 Reviews)

5 Star
100%
4 Star
0%
3 Star
0%
2 Star
0%
1 Star
0%

Have you used Recombinant Mouse IL-27 (NS0-expressed) Protein?

Submit a review and receive an Amazon gift card.

$25/€18/£15/$25CAN/¥75 Yuan/¥2500 Yen for a review with an image

$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image

Submit a Review

Filter by:


Recombinant Mouse IL-27 (NS0-expressed) Protein
By Anonymous on 03/17/2020
Application: Stem/Immune cell maintenance or differentiation
Reason for Rating: Good for Tr1 cell differentiation

Recombinant Mouse IL-27 (NS0-expressed) Protein
By Anonymous on 12/02/2019
Application: Immunoassay Standard

Recombinant Mouse IL-27 (NS0-expressed) Protein
By Anonymous on 06/15/2018
Application: Binding assay/Protein-protein interaction

Recombinant Mouse IL-27 (NS0-expressed) Protein
By Mahesh Raundhal on 04/13/2017
Application: T cell differentiation
Reason for Rating: Works well in inducing IFNg and IL-10 expression in naive mouse T cells.

Naive (CD4+CD44+CD62L++CD25-) mouse T cells were stimulated with anti-CD3, anti-CD28, IL-2 and 50 ng/mL IL-27. IFNg and IL-10 secretion was analyzed in the culture supernatants 5 days later by multiplex assays.