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#42022 Insulin/IGF-1 Signaling Pathway Antibody Sampler Kit

 
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2019年1月17日11時55分 現在
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#42022T1 Kit115,000
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キット内容 容量 用途 種交差性 ホモロジー† 検出分子量 アイソタイプ
Insulin Receptor β (4B8) Rabbit mAb #3025 20 µl WB, IP H, M, R 95 Rabbit IgG
IGF-I Receptor β (D23H3) XP® Rabbit mAb #9750 20 µl WB, IP, IF-IC, F H, M, R, Mk 95 Rabbit IgG
Phospho-IGF-I Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) Antibody #3021 20 µl WB, IP H, M, R B 95 Rabbit
Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 20 µl WB, IP, IHC-P, IF-IC, F H, M, R, Hm, Mk, Dm, Z, B C, X, Dg, Pg 60 Rabbit IgG
Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb #13038 20 µl WB, IP, IF-IC, F H, M, R, Mk 60 Rabbit IgG
Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb #5558 20 µl WB, IP, IF-IC, F H, M, R, Hm 46 Rabbit IgG
Phospho-FoxO1 (Thr24)/FoxO3a (Thr32)/FoxO4 (Thr28) (4G6) Rabbit mAb #2599 20 µl WB H, M, Mk 65, 78 to 82, 95 Rabbit
Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb #5536 20 µl WB, IP, IF-IC H, M, R, Mk R, C, Pg, Hr 289 Rabbit IgG
Phospho-Tuberin/TSC2 (Ser939) Antibody #3615 20 µl WB H, M 200 Rabbit
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl WB Goat

†Species predicted to react based on 100% sequence homology.

Applications Key: W=Western Blotting, IP=Immunoprecipitation, IF-IC=Immunofluorescence (Immunocytochemistry), F=Flow Cytometry, IHC-P=Immunohistochemistry (Paraffin)
Reactivity Key: H=Human, M=Mouse, R=Rat, Mk=Monkey, Hm=Hamster, Dm=D. melanogaster, Z=Zebrafish, B=Bovine

特異性・感度
Insulin Receptor beta (4B8) Rabbit mAb detects endogenous levels of total insulin receptor β. It does not cross-react with IGF-IR β. IGF-I Receptor β (D23H3) XP® Rabbit mAb detects endogenous levels of total IGF-I receptor β protein. This antibody does not cross-react with insulin receptor. Phospho-IGF-I Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) Antibody detects endogenous levels of Tyr1131-phosphorylated IGF-I receptor and Tyr1146-phosphorylated insulin receptor. The antibody cross-reacts with activated PDGF, FGF and EGF receptors, ErbB2 and c-Met. Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb detects endogenous levels of Akt only when phosphorylated at Ser473. Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb recognizes endogenous levels of Akt1 protein only when phosphorylated at Thr308. This antibody also recognizes endogenous levels of Akt2 protein when phosphorylated at Thr309 or Akt3 protein when phosphorylated at Thr305. Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb detects endogenous levels of GSK-3β only when phosphorylated at Ser9. This antibody reacts with denatured components of bovine serum, including BSA. Phospho-Tuberin/TSC2 (Ser939) Antibody detects endogenous levels of tuberin only when phosphorylated at serine 939. This antibody does not cross-react with tuberin phosphorylated at other sites. Phospho-FoxO1 (Thr24)/FoxO3a (Thr32)/Fox04 (Thr28) (4G6) Rabbit mAb detects endogenous levels of FoxO1 when phosphorylated at Thr24, of FoxO3a when phosphorylated at Thr32 or FoxO4 when phosphorylated at Thr28. Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb detects endogenous levels of mTOR protein only when phosphorylated at Ser2448.
使用抗原
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr999 of human insulin receptor β. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human IGF-I receptor β protein. Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues of human IGF-I Receptor β. Antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Ser473 of human Akt. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Thr308 of human Akt1 protein. Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser9 of human GSK-3β. Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Ser939 of human tuberin. Antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr28 of human Fox04. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser2448 of human mTOR protein.

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貯法
-20℃
社内データ

Western Blotting

Western Blotting

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.

Western Blotting

Western Blotting

Western blot analysis of extracts from 3T3-L1 adipocytes, untreated or insulin-treated (100 nM for the indicated times), using Phospho-IGF-I Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) Antibody (upper) or control IR antibody (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from NIH/3T3 cells, untreated, PDGF-treated, and PDGF and wortmannin-treated or PDGF and rapamycin-treated, using Phospho-Tuberin/TSC2 (Ser939) Antibody (top), Phospho-Tuberin/TSC2 (Thr1462) Antibody #3611 (middle) or Tuberin/TSC2 Antibody #3612 (bottom).


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Insulin Receptor β (4B8) Rabbit mAb.

Western Blotting

Western Blotting

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).

Western Blotting

Western Blotting

Western blot analysis of extracts from serum-starved NIH/3T3 cells, untreated or insulin-treated (150 nM, 5 minutes), alone or in combination with λ-phosphatase, using Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb (upper) or mTOR (7C10) Rabbit mAb #2983.


Western Blotting

Western Blotting

Western blot analysis of extracts from GSK-3α (-/-) (lanes 1,2) and GSK-3β (-/-) (lanes 3,4) mouse embryonic fibroblast (MEF) cells, λ phosphatase or PDGF-treated, using Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb (upper) and α/β-Tubulin Antibody #2148 (lower). (MEF wild type, GSK-3α (-/-) and GSK-3β (-/-) cells were kindly provided by Dr. Jim Woodgett, University of Toronto, Canada).

Western Blotting

Western Blotting

Western blot analysis of extracts from Jurkat cells treated with either Calyculin A (#9902) or LY294002 (#9901), NIH3T3 and COS-7 cells using Phospho-FoxO1 (Thr24)/(FoxO3a (Thr32)/FoxO4 (Thr28) (4G6) Rabbit mAb to detect FoxO1, FoxO3a and FoxO4 when phosphorylated at the Thr24, Thr32, and Thr28 positions, respectively (left panel). Total FoxO1, FoxO3a and FoxO4 were detected using FoxO1 (C29H4) Rabbit mAb (#2880), FoxO3a (75D8) Rabbit mAb (#2497) and FoxO4 Antibody (#9472), respectively (right panel).

Western Blotting

Western Blotting

Western blot analysis of extracts from 293 (IGF-I receptor β+) and SK-UT-1 (IGF-I receptor β-) cells using IGF-I Receptor β (D23H3) XP® Rabbit mAb (upper) or β-Actin Antibody #4967 (lower).


Western Blotting

Western Blotting

Western blot analysis of extracts from NIH/3T3 cells, untreated (-) or treated with Human Platelet-Derived Growth Factor AA (hPDGF-AA) #8913 (100 ng/ml, 5 min; +), and untreated (-) LNCaP and PC-3 cells, using Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from 293 cells, untreated or IGF-I-treated (100 nM for 2 minutes), using Phospho-IGF-I Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) Antibody (upper) or control IGF-I Receptor antibody (lower).

IHC-P (paraffin)

IHC-P (paraffin)

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.


IP

IP

Immunprecipitation of Insulin Receptor beta from insulin treated mIMCD-3 cell extracts using Insulin Receptor beta antibody (Lane 1) Lane 2: No antibody control. Lane 3: Input control.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, rapamycin-treated (#9904, 10 μM for 2 hours, left), insulin-treated (150 nM for 6 minutes, middle) or insulin- and λ-phosphatase-treated (right), using Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin. Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Western Blotting

Western Blotting

Western blot analysis of extracts from PC-3 cells, untreated or LY294002/wortmannin-treated, using Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb (upper) or GSK-3β (27C10) Rabbit mAb #9315 (lower).


Western Blotting

Western Blotting

Western blot analysis of extracts from Jurkat cells treated with either Calyculin A (#9902) or LY294002 (#9901) using Phospho-FoxO1 (Thr24)/(FoxO3a (Thr32)/FoxO4 (Thr28) (4G6) Rabbit mAb. The phospho-specificity of the antibody was verified by treating the membrane in the absence (-) or presence (-) of calf intestinal phosphatase (CIP) after western transfer.

IF-IC

IF-IC

Confocal immunofluorescent analysis of MCF7 (left) and SK-UT-1 (right) cells using IGF-I Receptor β (D23H3) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

IP

IP

Immunoprecipitation of phospho-Akt (Thr308) from Jurkat cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb.


IHC-P (paraffin)

IHC-P (paraffin)

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.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of NIH/3T3 cells, untreated (blue) or PDGF-treated (green), using Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of serum-starved NIH/3T3 cells, untreated (blue) or treated with Human Platelet-Derived Growth Factor AA (hPDGF-AA) #8913 (100 ng/ml, 15 min; green), using Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.


IHC-P (paraffin)

IHC-P (paraffin)

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

IF-IC

IF-IC

Confocal immunofluorescent analysis of wild type mouse embryonic fibroblasts (MEFs) (top row), GSK-3β (-/-) MEFs (middle row) , or PC-3 cells (bottom row), untreated (left), LY294002- and Wortmannin-treated (#9901 and #9951 respectively; center) or lambda phosphatase-treated (right), using Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). (MEF wild type and GSK-3β (-/-) cells were kindly provided by Dr. Jim Woodgett, University of Toronto, Canada).

IF-IC

IF-IC

Confocal immunofluorescent analysis of C2C12 cells, insulin-treated (100 nM, 15 min; left) or treated with LY294002 #9901 (50 μM, 2 hr; right), using Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).


IHC-P (paraffin)

IHC-P (paraffin)

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)).

IHC-P (paraffin)

IHC-P (paraffin)

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

IHC-P (paraffin)

IHC-P (paraffin)

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.)


IHC-P (paraffin)

IHC-P (paraffin)

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

Flow Cytometry

Flow Cytometry

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).

IF-IC

IF-IC

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).


バックグラウンド

Insulin and IGF-1 act on two closely related tyrosine kinase receptors to initiate a cascade of signaling events. These signaling events activate a variety of biological molecules, including kinases and transcription factors, which regulate cell growth, survival and metabolism.

Type I insulin-like growth factor receptor (IGF-IR) is a transmembrane receptor tyrosine kinase that is widely expressed in many cell lines and cell types within fetal and postnatal tissues (1-3). Three tyrosine residues within the kinase domain (Tyr1131, Tyr1135, and Tyr1136) are the earliest major autophosphorylation sites (4). Phosphorylation of these three tyrosine residues is necessary for kinase activation (5,6). Insulin receptors (IRs) share significant structural and functional similarity with IGF-I receptors, including the presence of an equivalent tyrosine cluster (Tyr1146/1150/1151) within the kinase domain activation loop. Tyrosine autophosphorylation of IRs is one of the earliest cellular responses to insulin stimulation (7). Autophosphorylation begins with phosphorylation at Tyr1146 and either Tyr1150 or Tyr1151, while full kinase activation requires triple tyrosine phosphorylation (8).

Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (9-11). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (10,11). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (12) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (13,14).

Tuberin is a product of the TSC2 tumor suppressor gene and an important regulator of cell proliferation and tumor development (15). Tuberin is phosphorylated on Ser939 and Thr1462 in response to PI3K activation and the human TSC complex is a direct biochemical target of the PI3K/Akt pathway (16). This result complements Drosophila genetics studies suggesting the possible involvement of the tuberin-hamartin complex in the PI3K/Akt mediated insulin pathway (17-19).

The mammalian target of rapamycin (mTOR, FRAP, RAFT) is a Ser/Thr protein kinase (20-22) that functions as an ATP and amino acid sensor to balance nutrient availability and cell growth (23,24). When sufficient nutrients are available, mTOR responds to a phosphatidic acid-mediated signal to transmit a positive signal to p70 S6 kinase and participate in the inactivation of the eIF4E inhibitor, 4E-BP1 (25). These events result in the translation of specific mRNA subpopulations. mTOR is phosphorylated at Ser2448 via the PI3 kinase/Akt signaling pathway and autophosphorylated at Ser2481 (26,27).

The Forkhead family of transcription factors is involved in tumorigenesis of rhabdomyosarcoma and acute leukemias (28-30). Within the family, three members (FoxO1, FoxO4, and FoxO3a) have sequence similarity to the nematode orthologue DAF-16, which mediates signaling via a pathway involving IGFR1, PI3K, and Akt (31-33). Active forkhead members act as tumor suppressors by promoting cell cycle arrest and apoptosis. Increased proliferation results when forkhead transcription factors are inactivated through phosphorylation by Akt at Thr24, Ser256, and Ser319, which results in nuclear export and inhibition of transcription factor activity (34).

Glycogen synthase kinase-3 (GSK-3) was initially identified as an enzyme that regulates glycogen synthesis in response to insulin (35). GSK-3 is a critical downstream element of the PI3K/Akt cell survival pathway whose activity can be inhibited by Akt-mediated phosphorylation at Ser21 of GSK-3α and Ser9 of GSK-3β (36,37).

  1. Adams, T.E. et al. (2000) Cell Mol Life Sci 57, 1050-93.
  2. Baserga, R. (2000) Oncogene 19, 5574-81.
  3. Scheidegger, K.J. et al. (2000) J Biol Chem 275, 38921-8.
  4. Hernández-Sánchez, C. et al. (1995) J Biol Chem 270, 29176-81.
  5. Lopaczynski, W. et al. (2000) Biochem Biophys Res Commun 279, 955-60.
  6. Baserga, R. (1999) Exp Cell Res 253, 1-6.
  7. White, M.F. et al. (1985) J Biol Chem 260, 9470-8.
  8. White, M.F. et al. (1988) J Biol Chem 263, 2969-80.
  9. Franke, T.F. et al. (1997) Cell 88, 435-7.
  10. Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.
  11. Franke, T.F. et al. (1995) Cell 81, 727-36.
  12. Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
  13. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
  14. Jacinto, E. et al. (2006) Cell 127, 125-37.
  15. Soucek, T. et al. (1998) Proc Natl Acad Sci U S A 95, 15653-8.
  16. Manning, B.D. et al. (2002) Mol Cell 10, 151-62.
  17. Gao, X. and Pan, D. (2001) Genes Dev 15, 1383-92.
  18. Potter, C.J. et al. (2001) Cell 105, 357-68.
  19. Tapon, N. et al. (2001) Cell 105, 345-55.
  20. Sabers, C.J. et al. (1995) J Biol Chem 270, 815-22.
  21. Brown, E.J. et al. (1994) Nature 369, 756-8.
  22. Sabatini, D.M. et al. (1994) Cell 78, 35-43.
  23. Gingras, A.C. et al. (2001) Genes Dev 15, 807-26.
  24. Dennis, P.B. et al. (2001) Science 294, 1102-5.
  25. Fang, Y. et al. (2001) Science 294, 1942-5.
  26. Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
  27. Peterson, R.T. et al. (2000) J Biol Chem 275, 7416-23.
  28. Anderson, M.J. et al. (1998) Genomics 47, 187-99.
  29. Galili, N. et al. (1993) Nat Genet 5, 230-5.
  30. Borkhardt, A. et al. (1997) Oncogene 14, 195-202.
  31. Nakae, J. et al. (1999) J Biol Chem 274, 15982-5.
  32. Rena, G. et al. (1999) J Biol Chem 274, 17179-83.
  33. Guo, S. et al. (1999) J Biol Chem 274, 17184-92.
  34. Arden, K.C. (2004) Mol Cell 14, 416-8.
  35. Welsh, G.I. et al. (1996) Trends Cell Biol 6, 274-9.
  36. Srivastava, A.K. and Pandey, S.K. (1998) Mol Cell Biochem 182, 135-41.
  37. Cross, D.A. et al. Nature 378, 785-9.
使用例
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関連製品
13038   Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb
2599   Phospho-FoxO1 (Thr24)/FoxO3a (Thr32)/FoxO4 (Thr28) (4G6) Rabbit mAb
3021   Phospho-IGF-I Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) Antibody
3025   Insulin Receptor β (4B8) Rabbit mAb
3615   Phospho-Tuberin/TSC2 (Ser939) Antibody
4060   Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb
5536   Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb
5558   Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb
7074   Anti-rabbit IgG, HRP-linked Antibody
9750   IGF-I Receptor β (D23H3) XP® Rabbit mAb

Tween is a registered trademark of ICI Americas, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
CST is a trademark of Cell Signaling Technology, Inc.
SignalStain is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.
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.

本製品は試験研究用です。

Insulin/IGF-1 Signaling Pathway Antibody Sampler Kit

Immune Cell Signaling Pathways

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