Revision 3

#36778Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, IF-F, IF-IC, FC-FP, ChIP

REACTIVITY:

H M R

SENSITIVITY:

Endogenous

MW (kDa):

60-80

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q96NM4, #O94900

Entrez-Gene Id:

84969, 9760

Product Information

Product Usage Information

For optimal ChIP results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 × 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
Immunofluorescence (Frozen) 1:100 - 1:400
Immunofluorescence (Immunocytochemistry) 1:100 - 1:400
Flow Cytometry (Fixed/Permeabilized) 1:200 - 1:800
Chromatin IP 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

For a carrier free (BSA and azide free) version of this product see product #86380.

Specificity / Sensitivity

Tox/Tox2 (E6G5O) Rabbit mAb recognizes endogenous levels of total Tox and Tox2 proteins. This antibody does not cross-react with Tox3 or Tox4 proteins. This antibody cross-reacts with an unidentified protein of 50 kDa in some murine cell extracts.

Species Reactivity:

Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gln435 of human Tox protein.

Background

Thymocyte selection-associated high mobility group box protein (Tox) is a DNA-binding nuclear factor and member of the evolutionarily conserved high-motility group (HMG)-box superfamily. Tox also defines a small subfamily of proteins that include Tox2, Tox3, and Tox4, all of which are highly conserved in vertebrate species but have unique tissue expression patterns and functions (1,2).

Tox plays a key role in T cell development in the thymus during positive selection (3-5). A study in Tox-deficient mice also revealed a requirement for Tox in CD4 T cell and NK cell lineage development, including NKT cells, FoxP3+ T regulatory T cells, and lymphoid tissue-inducer (LTi) cells (6-8). Although Tox expression is primarily restricted to developing immune cells in normal tissues, Tox is induced by high antigen stimulation during chronic viral infection or cancer, regulating T cell persistence and exhaustion (9-12). Tox has also been shown to be aberrantly expressed in cutaneous T cell lymphomas (13-14).

  1. O'Flaherty, E. and Kaye, J. (2003) BMC Genomics 4, 13.
  2. Aliahmad, P. et al. (2012) Curr Opin Immunol 24, 173-7.
  3. Wilkinson, B. et al. (2002) Nat Immunol 3, 272-80.
  4. Aliahmad, P. et al. (2004) J Exp Med 199, 1089-99.
  5. Chi, T.H. et al. (2002) Nature 418, 195-9.
  6. Aliahmad, P. and Kaye, J. (2008) J Exp Med 205, 245-56.
  7. Aliahmad, P. et al. (2010) Nat Immunol 11, 945-52.
  8. Yun, S. et al. (2011) Immunol Lett 136, 29-36.
  9. Page, N. et al. (2018) Immunity 48, 937-950.e8.
  10. Alfei, F. et al. (2019) Nature 571, 265-9.
  11. Yao, C. et al. (2019) Nat Immunol 20, 890-901.
  12. Wang, X. et al. (2019) J Hepatol pii: S0168-8278(19)30301-0. doi: 10.1016/j.jhep.2019.05.015.
  13. Morimura, S. et al. (2014) Arch Dermatol Res 306, 843-9.
  14. Huang, Y. et al. (2014) Oncotarget 5, 4418-25.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IF-F: Immunofluorescence (Frozen) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 3
#36778

Tox/Tox2 (E6G5O) Rabbit mAb

Western Blotting Image 1: Tox/Tox2 (E6G5O) Rabbit mAb Expand Image
Western blot analysis of extracts from various human cell lines using Tox/Tox2 (E6G5O) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 2: Tox/Tox2 (E6G5O) Rabbit mAb Expand Image
Western blot analysis of extracts from human, mouse, and rat tissues and mouse cell lines using Tox/Tox2 (E6G5O) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 3: Tox/Tox2 (E6G5O) Rabbit mAb Expand Image
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected (+) with constructs expressing Myc/DDK-tagged full-length human Tox protein (hTox-Myc/DDK), Myc/DDK-tagged full-length human Tox2 protein (hTox2-Myc/DDK), Myc/DDK-tagged full-length human Tox3 protein (hTox3-Myc/DDK), or Myc/DDK-tagged full-length human Tox4 protein (hTox4-Myc/DDK), using Tox/Tox2 (E6G5O) Rabbit mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunoprecipitation Image 1: Tox/Tox2 (E6G5O) Rabbit mAb Expand Image
Immunoprecipitation of Tox protein from SU-DHL-4 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Tox/Tox2 (E6G5O) Rabbit mAb. Western blot analysis was performed using Tox/Tox2 (E6G5O) Rabbit mAb.
Immunofluorescence Image 1: Tox/Tox2 (E6G5O) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of mouse thymus (left) and pancreas (right) using Tox/Tox2 (E6G5O) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Immunofluorescence Image 2: Tox/Tox2 (E6G5O) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of rat thymus (left) and pancreas (right) using Tox/Tox2 (E6G5O) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Immunofluorescence Image 3: Tox/Tox2 (E6G5O) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of mouse colon using Tox/Tox2 (E6G5O) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Immunofluorescence Image 1: Tox/Tox2 (E6G5O) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of SU-DHL-4 cells (left, positive) or A549 cells (right, negative) using Tox/Tox2 (E6G5O) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Flow Cytometry Image 1: Tox/Tox2 (E6G5O) Rabbit mAb Expand Image
Flow cytometric analysis of human peripheral blood mononuclear cells gated on lymphocytes co-stained with CD3 (UCHT1) Mouse mAb (APC Conjugate) #19881 using Tox/Tox2 (E6G5O) Rabbit mAb (right) compared to concentration matched

Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (left). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Flow Cytometry Image 2: Tox/Tox2 (E6G5O) Rabbit mAb Expand Image
Flow cytometric analysis of RPMI 8226 cells (blue, negative) and SU-DHL-4 cells (green, positive) using Tox/Tox2 (E6G5O) Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: Tox/Tox2 (E6G5O) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from untreated SU-DHL-4 cells and either Tox/Tox2 (E6G5O) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using human HERC2 intron 1 primers, human WAPAL exon 1 primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.