Revision 5
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-IC, FC-FP

REACTIVITY:

H M R

SENSITIVITY:

Endogenous

MW (kDa):

65, 75

Source/Isotype:

Rabbit IgG

UniProt ID:

#P17544, #P15336

Entrez-Gene Id:

11016, 1386

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
Immunofluorescence (Immunocytochemistry) 1:1600 - 1:6400
Flow Cytometry (Fixed/Permeabilized) 1:800 - 1:3200

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 #94125.

Specificity / Sensitivity

Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) (E6A8A) Rabbit mAb detects endogenous levels of ATF-2 only when dually phosphorylated at both Thr69 and Thr71, and ATF-7 only when dually phosphorylated at both Thr51 and Thr53. It does not recognize ATF-2 singly phosphorylated at either Thr69 or Thr71, and it does not recognize ATF-7 singly phosphorylated at either Thr51 or Thr53.

Species Reactivity:

Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr69 and Thr71 of human ATF-2 protein.

Background

The transcription factor ATF-2 (also called CRE-BP1) binds to both AP-1 and CRE DNA response elements and is a member of the ATF/CREB family of leucine zipper proteins (1). ATF-2 interacts with a variety of viral oncoproteins and cellular tumor suppressors and is a target of the SAPK/JNK and p38 MAP kinase signaling pathways (2-4). Various forms of cellular stress, including genotoxic agents, inflammatory cytokines, and UV irradiation, stimulate the transcriptional activity of ATF-2. Cellular stress activates ATF-2 by phosphorylation of Thr69 and Thr71 (2-4). Both SAPK and p38 MAPK have been shown to phosphorylate ATF-2 at these sites in vitro and in cells transfected with ATF-2. Mutations of these sites result in the loss of stress-induced transcription by ATF-2 (2-4). In addition, mutations at these sites reduce the ability of E1A and Rb to stimulate gene expression via ATF-2 (2).
ATF-7 is another member of the ATF/CREB family of leucine zipper proteins (5). Similarly, Thr51 and Thr53 (corresponding to Thr69 and Thr71 of ATF-2, respectively) can be phosphorylated under different conditions (6,7).

  1. Abdel-Hafiz, H.A. et al. (1992) Mol Endocrinol 6, 2079-89.
  2. Gupta, S. et al. (1995) Science 267, 389-93.
  3. van Dam, H. et al. (1995) EMBO J 14, 1798-811.
  4. Livingstone, C. et al. (1995) EMBO J 14, 1785-97.
  5. Peters, C.S. et al. (2001) J Biol Chem 276, 13718-26.
  6. Camuzeaux, B. et al. (2008) J Mol Biol 384, 980-91.
  7. Maekawa, T. et al. (2010) EMBO J 29, 196-208.

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 nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

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

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

使用に関する制限

法的な権限を与えられたCSTの担当者が署名した書面によって別途明示的に合意された場合を除き、 CST、その関連会社または代理店が提供する製品には以下の条件が適用されます。お客様が定める条件でここに定められた条件に含まれるものを超えるもの、 または、ここに定められた条件と異なるものは、法的な権限を与えられたCSTの担当者が別途書面にて受諾した場合を除き、拒絶され、 いかなる効力も効果も有しません。

研究専用 (For Research Use Only) またはこれに類似する表示がされた製品は、 いかなる目的についても FDA または外国もしくは国内のその他の規制機関により承認、認可または許可を受けていません。 お客様は製品を診断もしくは治療目的で使用してはならず、また、製品に表示された内容に違反する方法で使用してはなりません。 CST が販売または使用許諾する製品は、エンドユーザーであるお客様に対し、使途を研究および開発のみに限定して提供されるものです。 診断、予防もしくは治療目的で製品を使用することまたは製品を再販売 (単独であるか他の製品等の一部であるかを問いません) もしくはその他の商業的利用の目的で購入することについては、CST から別途許諾を得る必要があります。 お客様は以下の事項を遵守しなければなりません。(a) CST の製品 (単独であるか他の資材と一緒であるかを問いません) を販売、使用許諾、貸与、寄付もしくはその他の態様で第三者に譲渡したり使用させたりしてはなりません。また、商用の製品を製造するために CST の製品を使用してはなりません。(b) 複製、改変、リバースエンジニアリング、逆コンパイル、 分解または他の方法により製品の構造または技術を解明しようとしてはなりません。また、 CST の製品またはサービスと競合する製品またはサービスを開発する目的で CST の製品を使用してはなりません。(c) CST の製品の商標、商号、ロゴ、特許または著作権に関する通知または表示を除去したり改変したりしてはなりません。(d) CST の製品をCST 製品販売条件(CST’s Product Terms of Sale) および該当する書面のみに従って使用しなければなりません。(e) CST の製品に関連してお客様が使用する第三者の製品またはサービスに関する使用許諾条件、 サービス提供条件またはこれに類する合意事項を遵守しなければなりません。

Revision 5
#61584

Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) (E6A8A) Rabbit mAb

Western Blotting Image 1: Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) (E6A8A) Rabbit mAb Expand Image
Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated (-) or treated with Anisomycin #2222 (25 μg/ml, 30 min; +), using Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) (E6A8A) Rabbit mAb (upper), ATF-2/ATF-7 (A9G1M) Rabbit mAb #82870 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunoprecipitation Image 1: Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) (E6A8A) Rabbit mAb Expand Image
Immunoprecipitation of Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) protein from HeLa cell extracts treated with Anisomycin #2222 (25 μg/ml, 30 min). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) (E6A8A) Rabbit mAb. Western blot analysis was performed using Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) (E6A8A) Rabbit mAb.
Immunofluorescence Image 1: Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) (E6A8A) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HeLa cells either untreated (left), treated with Anisomycin #2222 (25 μg/mL, 30 min) (middle), or treated with anisomycin and post-processed with λ-phosphatase to confirm phospho-specificity (right) using Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) (E6A8A) Rabbit mAb (green) and Lamin A/C (4C11) Mouse mAb #4777 (red). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (pseudoblue).
Flow Cytometry Image 1: Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) (E6A8A) Rabbit mAb Expand Image
Flow cytometric analysis of Jurkat cells, untreated (blue, negative) or treated with Anisomycin #2222 (25 ug/mL, 30 min; green, positive) using Phospho-ATF-2 (Thr69/71)/ATF-7 (Thr51/53) (E6A8A) Rabbit mAb (solid lines) or 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.