Revision 3
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, W-S

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

40

Source/Isotype:

Rabbit IgG

UniProt ID:

#O75791

Entrez-Gene Id:

9402

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:50 - 1:250

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.

Specificity / Sensitivity

Phospho-GADS (Thr262) (E8L3B) Rabbit mAb recognizes endogenous levels of GADS protein only when phosphorylated at Thr262.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr262 of human GADS protein.

Background

GRB2-related adaptor downstream of Shc (GADS) belongs to the GRB2 family of adaptor proteins. It is a hematopoietic cell-specific signaling adaptor protein that harbors amino- and carboxy-terminal SH3 domains, a central SH2 domain, and a unique linker region that is rich in proline and glutamine residues (1). The presence of SH2 and SH3 domains within GADS strongly suggest that it functions in signal transduction cascades by facilitating protein-protein interactions. In the context of T cells, research studies have demonstrated that GADS interacts with LAT and SLP-76 signaling complexes to facilitate NFAT activation downstream of TCR engagement (2-4). Given its role as a fundamental mediator of TCR signaling, GADS is subject to multiple modes of negative regulation to limit TCR signal strength. For example, research studies have demonstrated that HPK1 directly phosphorylates GADS at Thr262 within its linker region, a modification that promotes 14-3-3 binding and dissociation of signaling complexes nucleated by LAT, SLP-76, and GADS (5). The linker region of GADS is also subject to caspase-mediated cleavage, which separates its SH2 and SH3 domains and thus impairs the ability of GADS to bridge LAT and SLP-76 signaling complexes for transduction of faithful TCR signals (6,7).

  1. Hámori, J. et al. (1978) Neuroscience 3, 403-12.
  2. Liu, S.K. et al. (1999) Curr Biol 9, 67-75.
  3. Law, C.L. et al. (1999) J Exp Med 189, 1243-53.
  4. Asada, H. et al. (1999) J Exp Med 189, 1383-90.
  5. Lasserre, R. et al. (2011) J Cell Biol 195, 839-53.
  6. Berry, D.M. et al. (2001) Oncogene 20, 1203-11.
  7. Yankee, T.M. et al. (2001) Proc Natl Acad Sci USA 98, 6789-93.

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 W-S: Simple Western™

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.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

限制使用

除非 CST 的合法授书代表以书面形式书行明确同意,否书以下条款适用于 CST、其关书方或分书商提供的书品。 任何书充本条款或与本条款不同的客书条款和条件,除非书 CST 的合法授书代表以书面形式书独接受, 否书均被拒书,并且无效。

专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品, (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Revision 3
#78972

Phospho-GADS (Thr262) (E8L3B) Rabbit mAb

Western Blotting Image 1: Phospho-GADS (Thr262) (E8L3B) Rabbit mAb Expand Image
使用 Phospho-GADS (Thr262) (E8L3B) Rabbit mAb(上图)、GADS (E4O1V) Rabbit mAb #95848(中图)或 β-Actin (D6A8) Rabbit mAb #8457(下图)对未经处理 (-) 或经 Rapid-Act T Cell Activation Kit (Human, Anti-CD3/CD28) #88179(6 分钟;+)处理且经血清饥饿的 Jurkat 细胞的提取物进行蛋白质印迹分析。
Western Blotting Image 2: Phospho-GADS (Thr262) (E8L3B) Rabbit mAb Expand Image
使用 Phospho-GADS (Thr262) (E8L3B) Rabbit mAb(上图)、GADS (E4O1V) Rabbit mAb #95848(中图)或 β-Actin (D6A8) Rabbit mAb #8457(下图)对未经处理 (-) 或经 H2O2(11 mM,3 分钟;+)处理且经血清饥饿的 Jurkat 细胞的提取物进行蛋白质印迹分析。如图所示,经 H2O2 处理的裂解物用牛小肠磷酸酶和 λ 磷酸酶(CIP/λ 磷酸酶;+)处理。
Western Blotting Image 1: Phospho-GADS (Thr262) (E8L3B) Rabbit mAb Expand Image
Simple Western™ analysis of lysates (1.0 mg/mL) from serum starved Jurkat cells treated with anti-human CD3 (10ug/mL, 15min) and anti-human CD28 (10ug/mL, 15min), then goat anti-mouse IgG (20ug/mL, 15min) using Phospho-GADS (Thr262) (E8L3B) Rabbit mAb #78972. 虚拟泳道式图像(左图)显示一抗稀释比例为 1:50 和 1:250 时的靶标条带(如图所示)。对应的电泳图(右图)为一抗稀释比例在 1:50(蓝线)和 1:250(绿线)时沿毛细血管内分子量的化学发光结果。在还原条件下,使用 12-230 kDa 分离模块在 ProteinSimple(BioTechne 品牌)的 Jess™ Simple Western 仪器上进行该实验。