Revision 11
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, IP, IHC-Bond, IHC-P, IF-IC, FC-FP, ChIP, ChIP-seq

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

79, 86

Source/Isotype:

Rabbit IgG

UniProt ID:

#P40763

Entrez-Gene Id:

6774

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 5 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

Application Dilution
Western Blotting 1:2000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:100
IHC Leica Bond 1:100 - 1:400
Immunohistochemistry (Paraffin) 1:100 - 1:400
Immunofluorescence (Immunocytochemistry) 1:100 - 1:200
Flow Cytometry (Fixed/Permeabilized) 1:100 - 1:400
Chromatin IP 1:100
Chromatin IP-seq 1:100

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 #73533.

Specificity / Sensitivity

Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb detects endogenous levels of Stat3 only when phosphorylated at tyrosine 705. This antibody does not cross-react with phospho-EGFR or the corresponding phospho-tyrosines of other Stat proteins.

Species Reactivity:

Human, Mouse, Rat, Monkey

Species predicted to react based on 100% sequence homology

Hamster, Bovine, Pig, Horse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr705 of mouse Stat3.

Background

The Stat3 transcription factor is an important signaling molecule for many cytokines and growth factor receptors (1) and is required for murine fetal development (2). Research studies have shown that Stat3 is constitutively activated in a number of human tumors (3,4) and possesses oncogenic potential (5) and anti-apoptotic activities (3). Stat3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding (6,7). Transcriptional activation seems to be regulated by phosphorylation at Ser727 through the MAPK or mTOR pathways (8,9). Stat3 isoform expression appears to reflect biological function as the relative expression levels of Stat3α (86 kDa) and Stat3β (79 kDa) depend on cell type, ligand exposure, or cell maturation stage (10). It is notable that Stat3β lacks the serine phosphorylation site within the carboxy-terminal transcriptional activation domain (8).

  1. Heim, M.H. (2001) J Recept Signal Transduct Res 19, 75-120.
  2. Takeda, K. et al. (1997) Proc Natl Acad Sci U S A 94, 3801-4.
  3. Catlett-Falcone, R. et al. (1999) Immunity 10, 105-15.
  4. Garcia, R. and Jove, R. (1998) J Biomed Sci 5, 79-85.
  5. Bromberg, J.F. et al. (1999) Cell 98, 295-303.
  6. Darnell, J.E. et al. (1994) Science 264, 1415-21.
  7. Ihle, J.N. (1995) Nature 377, 591-4.
  8. Wen, Z. et al. (1995) Cell 82, 241-50.
  9. Yokogami, K. et al. (2000) Curr Biol 10, 47-50.
  10. Biethahn, S. et al. (1999) Exp Hematol 27, 885-94.

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™ IP: Immunoprecipitation IHC-Bond: IHC Leica Bond IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq

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.
XP is a registered 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

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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 11
#9145

Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb

Western Blotting Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
使用 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb #9145(上图)、Stat3 (D3Z2G) Rabbit mAb #12640(中图)和 β-Actin (D6A8) Rabbit mAb #8457(下图)对未经处理或已经过 IFNa(#36000,100 ng/mL,5 分钟)或 IFNg(#80385,100 ng/mL,30 分钟)处理的 HeLa 细胞的提取物进行蛋白质印迹法分析。
Western Blotting Image 2: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
使用 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb,对 IFN-α 处理的 Jurkat 细胞、Hela 细胞(左)、经 EGF 处理的 A431 细胞(右)提取物进行蛋白质印迹分析。注意,抗体可检测 A431 的基础磷酸化 Stat3。
Western Blotting Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
使用 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb #9145 对经过 IFN-α(100 ng/mL,5 分钟)处理的血清饥饿 HeLa 细胞的裂解物 (0.1 mg/mL) 进行 Simple Western™ 分析。虚拟泳道式图像(左图)显示一抗稀释比例为 1:10 和 1:50 时的单一靶标条带(如图所示)。对应的电泳图(右图)为一抗稀释比例在 1:10(蓝线)和 1:50(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。
Immunoprecipitation Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
对经人 IFN-α(50 ng/ml,15 分钟)处理的 U266 提取物中的磷酸化 Stat3 (Tyr705) 进行免疫沉淀分析。泳道 1 为 10% input,泳道 2 为 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900,泳道 3 为 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb。使用 Phospho-Stat3 (Tyr705) (3E2) Mouse mAb #9138 进行蛋白质印迹法分析。Anti-mouse IgG, HRP-linked Antibody #7076 用作二抗。
Immunohistochemistry Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
在 Leica® BOND Rx 上使用 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb 对石蜡包埋的人结肠腺癌细胞进行免疫组织化学分析。
Immunohistochemistry Image 2: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
在 Leica® BOND Rx 上使用 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb 对石蜡包埋的人肺神经内分泌癌细胞进行免疫组织化学分析。
Immunohistochemistry Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
使用 Phosho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb 对石蜡包埋的 Apc (min/+) 小鼠肠道进行免疫组织化学分析。
Immunohistochemistry Image 2: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
使用 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb 对石蜡包埋的人肺癌进行免疫组织化学分析,显示出胞核定位。
Immunohistochemistry Image 3: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
使用 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb,对未经处理(左)或经 λ 磷酸酶处理(右)的石蜡包埋的人乳腺癌(尤其是内皮细胞)进行免疫组织化学分析。
Immunohistochemistry Image 4: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
使用 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb 在 SignalSlide® HeLa -/+ IFNa IHC Controls #55861 上(对未经处理(左图)或经 Human Interferon-α1 (hIFN-α1) #8927 处理(右图)的石蜡包埋的 Hela 细胞沉淀物)进行免疫组织化学分析。
Immunofluorescence Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
对 IFN-α 处理(左)或未经处理(右)Hela 细胞进行共聚焦免疫荧光分析,用 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb(绿色)标记。
Flow Cytometry Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
使用 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb(实线)或浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线)对未经处理(蓝色)或已经过 IFNα(50 ng/ml,15 分钟;绿色)处理的 U266 细胞进行流式细胞分析。Anti-rabbit IgG (H+L)、F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 作为二抗。
Chromatin Immunoprecipitation Image 1: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对饥饿过夜后,用 IL-6(100 ng/ml;30 分钟)处理的 Hep G2 细胞中提取的交联染色质,在加入 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb 后,进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在 IRF1 内结合,IRF1 是 Phospho-Sata3 的一种已知靶标基因(参见包含 ChIP-qPCR 数据的其他结果图)。欲知其他 ChIP-seq 情况,请下载产品说明书。
Chromatin Immunoprecipitation Image 2: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
使用 SimpleChIP® Plus Sonication Chromatin IP Kit #56383,对饥饿过夜后,用 IL-6(100 ng/ml)处理30 分钟)的 Hep G2 细胞中提取的交联染色质,在加入 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb 或 Normal Rabbit IgG #2729 后,进行染色质免疫沉淀分析。使用 SimpleChIP® Human c-Fos Promoter Primers #4663、human IRF1 promoter primers 和 SimpleChIP® Human α Satellite Repeat Primers #4486,通过实时 PCR 对富集的 DNA 进行量化。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。
Chromatin Immunoprecipitation Image 3: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对饥饿过夜后,用 IL-6(100 ng/ml;30 分钟)处理的 Hep G2 细胞中提取的交联染色质,在加入 Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb 或 Normal Rabbit IgG #2729 后,进行染色质免疫沉淀。使用 human IRF-1 promoter primers、SimpleChIP® Human c-Fos Promoter Primers #4663 和 SimpleChIP® Human α Satellite Repeat Primers #4486 并通过实时 PCR 对富集的 DNA 进行定量分析。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。