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9918
Translational Control Antibody Sampler Kit

Translational Control Antibody Sampler Kit #9918

Western Blotting Image 1

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 2

Western blot analysis of extracts from serum starved or serum treated (20%) 293, NIH/3T3, and PC12 cells, using Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb (upper), or p70 S6 Kinase (49D7) rabbit mAb #2708 (lower).

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Western Blotting Image 3

Western blot analysis of extracts from PC12 and NIH/3T3 cells, treated with λ phosphatase, 20% FBS (20 min) or 100 ng/ml PDGF (20 min) as indicated, using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb (upper) or S6 Ribosomal Protein (5G10) Rabbit mAb #2217 (lower).

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Western Blotting Image 4

Western blot analysis of extracts from C2C12 cells, untreated or thapsigargin-treated, using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb (upper) or eIF2α Antibody #9722 (lower).

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Western Blotting Image 5

Western blot analysis of extracts from 293T cells using 4E-BP1 Antibody #9452 (upper) and Phospho-4E-BP1 (Thr37/46) Antibody #2855 (lower). The cells were starved for 24 hours in serum-free medium and underwent a 1 hour amino acid deprivation. Amino acids were replenished for 1 hour. Cells were then either untreated (-) or treated with 100 nM insulin (+) for 30 minutes.

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Western Blotting Image 6

Western blot analysis of extracts from NIH/3T3 cells, untreated or treated with serum, PD98059 or Dexamethasone, using Phospho-eIF4E (Ser209) Antibody (upper) or eIF4E Antibody #9742 (lower).

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Western Blotting Image 7

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 8

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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IHC-P (paraffin) Image 9

Immunohistochemical analysis using Phospho-S6 Ribosomal Protein (S235/236) (D57.2.2E) 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-P (paraffin) Image 10

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, untreated (left) or λ phosphatase-treated (right), using Phopsho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb.

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IHC-P (paraffin) Image 11

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb.

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IHC-P (paraffin) Image 12

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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IHC-P (paraffin) Image 13

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb in the presence of control peptide (left) or Phospho-S6 Ribosomal Protein (Ser235/236) Blocking Peptide #1220 (right).

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IHC-P (paraffin) Image 14

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb.

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IHC-P (paraffin) Image 15

Immunohistochemical analysis of paraffin-embedded human lymphoma using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb.

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IHC-P (paraffin) Image 16

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

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IHC-P (paraffin) Image 17

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb.

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IHC-P (paraffin) Image 18

Immunohistochemical analysis of paraffin-embedded human lymphoma using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb.

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IHC-P (paraffin) Image 19

Immunohistochemical analysis using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb on SignalSlide (TM) Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells untreated (left) or LY294002-treated (right)).

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IHC-P (paraffin) Image 20

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-P (paraffin) Image 21

Immunohistochemical analysis of paraffin-embedded LNCaP cells, untreated (left) or rapamycin-treated (right), using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb.

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IHC-P (paraffin) Image 22

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb in the presence of control peptide (left) or Phospho-4E-BP1 (Thr37/46) Blocking Peptide #1052 (right).

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IHC-P (paraffin) Image 23

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

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IHC-P (paraffin) Image 24

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb.

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Flow Cytometry Image 25

Flow cytometric analysis of Jurkat cells, untreated (green) or LY294002, Wortmannin and U0126-treated (blue), using Phospho-4E-BP1 (Thr36/46) (236B4) Rabbit mAb compared to a nonspecific negative control antibody (red).

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IHC-P (paraffin) Image 26

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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IHC-P (paraffin) Image 27

Immunohistochemical analysis of paraffin-embedded mouse spleen using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb.

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IF-IC Image 28

Confocal immunofluorescent analysis of 293 cells, expressing either non-targeting shRNA (top) or shRNA targeting 4E-BP1/2 (bottom), using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (green). To confirm phospho-specificity, cells were treated with an inhibitor cocktail consisting of LY294002 #9901, U0126 #9903, and Rapamycin #9904 (50 μM; 10 μm; 10 nM; 2 hr) (left), stimulated with insulin (100 nM, 30 min; middle), or processed with λ-phosphatase following insulin stimulation (right). Red = Propidium Iodide (PI)/RNase Staining Solution (#4087).

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IHC-P (paraffin) Image 29

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-P (paraffin) Image 30

Immunohistochemical analysis of paraffin-embedded A549 xenograft, untreated (left) or λ phosphatase-treated (right), using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb.

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IHC-F (frozen) Image 31

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

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IHC-F (frozen) Image 32

Immunohistochemical analysis of frozen U-87MG xenograft using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb.

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Flow Cytometry Image 33

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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Flow Cytometry Image 34

Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002, wortmannin and U0126 (blue), using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb compared to a nonspecific negative control antibody (red).

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IF-IC Image 35

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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IF-IC Image 36

Confocal immunofluorescent analysis of HeLa cells, rapamycin-treated (left) or 20% serum-treated (right), using Phospho-S6 Ribosomal protein (Ser235/Ser236) (D57.2.2E) XP® Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
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-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb 9234 20 µl
  • WB
H M R Mk 70, 85 Rabbit IgG
Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb 4858 20 µl
  • WB
  • IHC
  • IF
  • F
H M R Mk Mi Sc 32 Rabbit IgG
Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb 3398 20 µl
  • WB
  • IP
  • IHC
H M R Mk Dm 38 Rabbit IgG
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb 2855 20 µl
  • WB
  • IHC
  • IF
  • F
H M R Mk Dm 15 to 20 Rabbit IgG
Phospho-eIF4E (Ser209) Antibody 9741 20 µl
  • WB
H M R Mk 25 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Translational Control Antibody Sampler Kit provides a fast and economical means of evaluating multiple proteins involved in translational control. The kit contains enough primary and secondary antibody to perform two Western blot experiments.

Each phospho-specific antibody in the Translational Control Antibody Sampler Kit detects the intended target only when phosphorylated at the indicated site. Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb may cross-react with 4E-BP2 and 4E-BP3 when phosphorylated at equivalent sites.

Phospho-specific polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser209 of human elF4E. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Phospho-specific rabbit monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser473 of mouse Akt, Thr37 and Thr46 of mouse 4E-BP1, Ser51 of human eIF2α, Ser235 and Ser236 of human ribosomal protein S6, and Thr389 of human p70 S6 kinase.

Key steps in translational control occur at the level of eukaryotic initiation factor 4F (eIF4F) and p70 S6 kinase regulation. eIF4F is a complex whose functions include the recognition of the mRNA 5' cap structure. Several stimuli, such as insulin and various growth and survival factors, regulate the eIF4F complex and p70 S6 kinase primarily by triggering a signaling cascade dependent on sequential activation of PI3K, Akt/PKB and mTOR/FRAP kinases. Akt is activated by phosphorylation within the C-terminus at Ser473 and within the activation loop at Thr308 by phospholipid-dependent kinases. Inactivation in vivo of PI3K by the highly selective inhibitor LY294002 inhibits Akt and downstream elements of this cascade. Direct phosphorylation of mTOR/FRAP at Ser2448 by Akt is a key regulatory event controlling its kinase activity. mTOR/FRAP activity can be effectively blocked by Rapamycin, leading to inactivation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1), an inhibitor of translation initiation, and activation of p70 S6 kinases. Inactivation of 4E-BP1 by sequential phosphorylation causes the release of eIF4E, which, together with eIF4G and other factors, forms a functional eIF4F cap binding complex. p70 S6 kinases phosphorylates the 40S ribosomal subunit protein S6 and stimulates the translation of 5' oligopyrimidine tract containing mRNAs. The Erk pathway is also involved in regulation at this level by regulating the eIF4E kinase, Mnk1, and activating p70 S6 kinase. Tuberin, a product of the tumor supressor gene TSG2, is directly phosphorylated atThr1462 by Akt/PKB. Tuberin inhibits the mammalian target of rapamycin, mTOR, which results in inhibition of p70 S6 kinase and activation of 4E-BP1 and, therefore, inhibition of translation.

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  2. Gingras, A.C. et al. (2001) Genes Dev 15, 807-26.
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For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.