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8336
ALK Activation Antibody Sampler Kit

ALK Activation Antibody Sampler Kit #8336

Western Blotting Image 1

Western blot analysis of Sup-M2 cell lysate, using Phospho-ALK (Tyr1586) (3B4) Rabbit mAb (A and B), and ALK Antibody (C and D).The phospho-specificity of this antibody was characterized by treating the membrane with calf intestinal alkaline phosphatase (CIP) (B and D) after Western transfer.

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Western Blotting Image 2

Western blot analysis of extracts from NK-92 cells, untreated or treated with Human Interleukin-2 (hIL-2) #8907 (10 ng/ml, 15 minutes), using Phospho-Jak3 (Tyr980/981) (D44E3) Rabbit mAb (upper) or Jak3 Antibody #3775 (lower).

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Western Blotting Image 3

Western blot analysis of extracts from UT-7 cells, untreated or treated with erythropoietin (EPO; 3 units/ml; 5 min) and from untreated HEL cells, using Phospho-Jak2 (Tyr1007) (D15E2) Rabbit mAb (upper) or total Jak2 (D2E12) Rabbit mAb #3230 (lower). HEL cells contain a V617F activating mutation in Jak2.

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Western Blotting Image 4

Western blot analysis of extracts from IFN-alpha treated Jurkat cells and HeLa cells (left), as well as EGF treated A431 cells (right), using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb. Note that the basal phospho-Stat3 in A431 is detected by the antibody.

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Chromatin IP-seq Image 5

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hep G2 cells starved overnight and treated with IL-6 (100 ng/ml) for 30 minutes and Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across IRF1, a known target gene of Phospho-Sata3 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

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Western Blotting Image 6

Western blot analysis of extracts from UT-7 cells, untreated or treated with erythropoietin (EPO; 3 units/ml for 5 min), TF-1 cells, untreated or treated with Human Granulocyte Macrophage Colony Stimulating Factor #8922 (hGM-CSF; 100 ng/ml for 10 min), and NK-92 cells, untreated or treated with Human Interleukin-2 #8907 (hIL-2; 100 ng/ml for 10 min), using Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb.

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Western Blotting Image 7

Western blot analysis of extracts from COS cells, untreated or treated with either U0126 #9903 (10 µM for 1h) or TPA #4174 (200 nM for 10 m), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 and p44/42 MAPK (Erk1/2) (3A7) Mouse mAb #9107.

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Western Blotting Image 8

Western blot analysis of extracts from PC-3 cells, untreated or LY294002/wortmannin-treated, and NIH/3T3 cells, serum-starved or PDGF-treated, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

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Western Blotting Image 9

Western blot analysis of extracts NIH/3T3 cells, serum-starved or treated with human Platelet-Derived Growth Factor BB hPDGF-BB #8912 (100 ng/ml, 15 min), using Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb (upper) or Src (36D10) Rabbit mAb #2109 (lower).

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Western Blotting Image 10

Western blot analysis of extracts from NIH/3T3 cells, untreated (-) or stimulated with hPDGF-BB #8912 (5 min; +), and from A-431 cells, untreated (-) or stimulated with hEGF #8916 (5 min; +), using Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 11

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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IHC-P (paraffin) Image 12

Immunohistochemical analysis of paraffin-embedded Apc (min/+) mouse intestine, using Phosho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

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Chromatin IP Image 13

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hep G2 cells starved overnight and treated with IL-6 (100 ng/ml) for 30 minutes, and either Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb or of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human IRF-1 promoter primers, SimpleChIP® Human c-Fos Promoter Primers #4663, 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.

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Flow Cytometry Image 14

Flow cytometric analysis of TF-1 cells, untreated (blue) or GM-CSF treated (green), using Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb.

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Western Blotting Image 15

Western blot analysis of extracts from 293, NIH/3T3 and C6 cells, treated with λ phosphatase or TPA #4174 as indicated, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (upper), or p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695 (lower).

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IHC-P (paraffin) Image 16

Immunohistochemical analysis of paraffin-embedded MDA-MB-468 xenograft using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (left) or PTEN (138G6) Rabbit mAb #9559 (right). Note the presence of P-Akt staining in the PTEN deficient MDA-MB-468 cells.

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IP Image 17

Immunoprecipitation of phospho-PLCγ1 (Tyr783) from A-431 cell extracts stimulated with hEGF #8916 using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb.

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IHC-P (paraffin) Image 18

Immunohistochemical analysis of paraffin embedded human breast carcinoma, specifically endothelial cells, untreated (left) or lambda phosphatase treated (right), using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

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IF-IC Image 19

Confocal immunofluorescent analysis of A-431 cells, EGF-treated (left) or untreated (right), using Phospho-Stat5 (Tyr694) XP®(D47E7) Rabbit mAb (green) and Pan-Keratin (C11) Mouse mAb #4545 (red).

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IHC-P (paraffin) Image 20

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb.

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IHC-P (paraffin) Image 21

Immunohistochemical analysis of paraffin-embedded human breast carcinoma comparing SignalStain® Antibody Diluent #8112 (left) to TBST/5% normal goat serum (right) using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060.

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Flow Cytometry Image 22

Flow cytometric analysis of NIH/3T3 cells, untreated (blue) or treated with recombinant mouse PDGF-BB (200 ng/ml, 15 min; green), using Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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IHC-P (paraffin) Image 23

Immunnohistochemical analysis of paraffin-embedded human lung carcinoma, showing nuclear localization, using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

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IHC-P (paraffin) Image 24

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, untreated (left) or λ phosphatase-treated (right), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb.

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IHC-P (paraffin) Image 25

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

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IHC-P (paraffin) Image 26

Immunohistochemical analysis using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb on SignalSlide® HeLa -/+ IFNa IHC Controls #55861 (paraffin-embedded HeLa cell pellets, untreated (left) or treated with Human Interferon-α1 (hIFN-α1) #8927 (right)).

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IHC-P (paraffin) Image 27

Immunohistochemical analysis using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb on SignalSlide™ Phospho-p44/42 MAPK (Thr202/Tyr204) IHC Controls #8103 (paraffin-embedded NIH/3T3 cells, treated with U0126 #9903 (left) or TPA #4174 (right).

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IHC-P (paraffin) Image 28

Immunohistochemical analysis using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb on SignalSlide® Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right)).

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IHC-F (frozen) Image 29

Immunohistochemical analysis of frozen H1650 xenograft using Phospho-Stat3 (Tyr705)(D3A7) XP® Rabbit mAb.

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Flow Cytometry Image 30

Flow cytometric analysis of Jurkat cells, treated with U0126 (10 µM, 2 hrs; green) or treated with TPA #4174 (200 nM, 30 min; blue) using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® 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.

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IHC-P (paraffin) Image 31

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

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Flow Cytometry Image 32

Flow cytometric analysis of HeLa cells, untreated (blue) or IFN-α treated (green), using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

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IF-IC Image 33

Confocal immunofluorescent analysis of Drosophila egg chambers, untreated (top) or λ phosphatase-treated (bottom), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (green) and S6 Ribosomal Protein (54D2) Mouse mAb #2317 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 34

Immunohistochemical analysis of paraffin-embedded PTEN heterozygous mutant mouse endometrium using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb. (Tissue section courtesy of Dr. Sabina Signoretti, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.)

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IF-IC Image 35

Confocal immunofluorescent analysis of HeLa cells, IFN-alpha treated (left) or untreated (right), labeled with Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb (green).

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IF-IC Image 36

Confocal immunofluorescent analysis of HT1080 cells, starved overnight then treated with U0126 #9903 (10 uM, 2 h; left) or PDBu (Phorbol 12,13-Dibutyrate) #12808 (100 nM, 15 m; right) using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 37

Immunohistochemical analysis of paraffin-embedded U-87MG xenograft, untreated (left) or lambda phosphatase-treated (right), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

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IHC-F (frozen) Image 38

Immunohistochemical analysis of frozen SKOV3 xenograft using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

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Flow Cytometry Image 39

Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, wortmannin #9951 and U0126 #9903 (blue), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb compared to a nonspecific negative control antibody (red).

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IF-IC Image 40

Confocal immunofluorescent analysis of C2C12 cells, LY294002-treated (left) or insulin-treated (right), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin #8953 (red). Blue pseudocolor = DRAQ5®#4084 (fluorescent DNA dye).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-ALK (Tyr1586) (3B4) Rabbit mAb 3348 20 µl
  • WB
H 80 (NPM-ALK) 220 (ALK) Rabbit IgG
Phospho-Jak3 (Tyr980/981) (D44E3) Rabbit mAb 5031 20 µl
  • WB
H M 115 Rabbit IgG
Phospho-Jak2 (Tyr1007) (D15E2) Rabbit mAb 4406 20 µl
  • WB
H M 125 Rabbit IgG
Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb 9145 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H M R Mk 79, 86 Rabbit IgG
Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb 4322 20 µl
  • WB
  • IF
  • F
H M 90 Rabbit IgG
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb 4370 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Hm Mk Mi Dm Z B Dg Pg Sc 44, 42 Rabbit IgG
Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb 4060 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Hm Mk Dm Z B 60 Rabbit IgG
Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb 6943 20 µl
  • WB
  • IP
H M R Mk 60 Rabbit IgG
Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb 14008 20 µl
  • WB
  • IP
  • F
H M 155 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The ALK Activation Antibody Sampler Kit provides an economical means to evaluate the activation status of multiple members of the ALK pathway, including phosphorylated ALK, Jak2, Jak3, Stat3, Stat5, PLCγ1, Akt, Src, and p44/42 MAPK. The kit includes enough antibody to perform two western blot experiments with each primary antibody.

Each antibody in the ALK Activation Antibody Sampler Kit recognizes the phosphorylated form of its specific target. Phospho-ALK (Tyr1586) (3B4) Rabbit mAb may cross-react with other activated protein tyrosine kinases including EGFR. Phospho-Jak2 (Tyr1007) (D15E2) Rabbit mAb may cross react with phosphorylated Jak3 and Tyk2. Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb may cross react with overexpressed phosphorylated RTKs.

Rabbit monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Tyr1586 of human ALK, Tyr1007 of human Jak2, Tyr980/981 of human and mouse Jak3, Tyr705 of mouse Stat3, Tyr694 of human Stat5a, Thr202/Tyr204 of human p44 MAP kinase, Ser473 of human Akt, Tyr416 of human Src, or Tyr783 of human PLCγ1 protein.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor for pleiotrophin (PTN), a growth factor involved in embryonic brain development (1-3). In ALK-expressing cells, PTN induces phosphorylation of both ALK and the downstream effectors IRS-1, Shc, PLCγ, and PI3 kinase (1). ALK was originally discovered as a nucleophosmin (NPM)-ALK fusion protein produced by a translocation (4). Investigators have found that the NPM-ALK fusion protein is a constitutively active, oncogenic tyrosine kinase associated with anaplastic lymphoma (4). Research literature suggests that activation of PLCγ by NPM-ALK may be a crucial step for its mitogenic activity and involved in the pathogenesis of anaplastic lymphomas (5).

A distinct ALK oncogenic fusion protein involving ALK and echinoderm microtubule-associated protein like 4 (EML4) has been described in the research literature from a non-small cell lung cancer (NSCLC) cell line, with corresponding fusion transcripts present in some cases of lung adenocarcinoma. The short, amino-terminal region of the microtubule-associated protein EML4 is fused to the kinase domain of ALK (6-8).

  1. Stoica, G.E. et al. (2001) J Biol Chem 276, 16772-9.
  2. Iwahara, T. et al. (1997) Oncogene 14, 439-49.
  3. Morris, S.W. et al. (1997) Oncogene 14, 2175-88.
  4. Morris, S.W. et al. (1994) Science 263, 1281-4.
  5. Bai, R.Y. et al. (1998) Mol Cell Biol 18, 6951-61.
  6. Rikova, K. et al. (2007) Cell 131, 1190-203.
  7. Takeuchi, K. et al. (2008) Clin Cancer Res 14, 6618-24.
  8. Soda, M. et al. (2007) Nature 448, 561-6.
Entrez-Gene Id
207 , 208 , 10000 , 238 , 5595 , 5594 , 2534 , 3055 , 3717 , 3718 , 3932 , 4067 , 5335 , 6714 , 6774 , 6776 , 6777 , 7525
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Select antibodies in this kit are sold under a license from Chemicon International, Inc. relating to U.S. Patent No. 5,658,791.

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