Revision 1

#9946Store at -20C

1 Kit

(8 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Phospho-FoxO1 (Thr24)/FoxO3a (Thr32) Antibody 9464 20 µl 78 to 82, 95 kDa Rabbit 
Phospho-FoxO1 (Ser256) Antibody 9461 20 µl 82 kDa Rabbit 
Phospho-FoxO1 (Thr24)/FoxO3a (Thr32)/FoxO4 (Thr28) (4G6) Rabbit mAb 2599 20 µl 65, 78 to 82, 95 kDa Rabbit 
FoxO1 (C29H4) Rabbit mAb 2880 20 µl 78 to 82 kDa Rabbit IgG
Phospho-FoxO3a (Ser253) (D18H8) Rabbit mAb 13129 20 µl 97 kDa Rabbit IgG
Phospho-FoxO3a (Ser318/321) Antibody 9465 20 µl 97 kDa Rabbit 
FoxO4 Antibody 9472 20 µl 65 kDa Rabbit 
FoxO3a (D19A7) Rabbit mAb 12829 20 µl 82 to 97 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

This sampler kit provides an economical means to investigate Forkhead signaling. The kit contains primary and secondary antibodies to perform two Western blots with each antibody.

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.

Background

The Forkhead family of transcription factors is involved in tumorigenesis of rhabdomyosarcoma and acute leukemias (1-3). 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 (4-6). Active forkhead members act as tumor suppressors by promoting cell cycle arrest and apoptosis. Increased expression of any FoxO member results in the activation of the cell cycle inhibitor p27 Kip1. Forkhead transcription factors also play a part in TGF-β-mediated upregulation of p21 Cip1, a process negatively regulated through PI3K (7). 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 (8). Forkhead transcription factors can also be inhibited by the deacetylase sirtuin (SirT1) (9).

  1. Anderson, M.J. et al. (1998) Genomics 47, 187-99.
  2. Galili, N. et al. (1993) Nat Genet 5, 230-5.
  3. Borkhardt, A. et al. (1997) Oncogene 14, 195-202.
  4. Nakae, J. et al. (1999) J Biol Chem 274, 15982-5.
  5. Rena, G. et al. (1999) J Biol Chem 274, 17179-83.
  6. Guo, S. et al. (1999) J Biol Chem 274, 17184-92.
  7. Seoane, J. et al. (2004) Cell 117, 211-23.
  8. Arden, K.C. (2004) Mol Cell 14, 416-8.
  9. Yang, Y. et al. (2005) EMBO J 24, 1021-32.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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    Revision 1
    #9946

    Forkhead Signaling Antibody Sampler Kit

    Forkhead Signaling Antibody Sampler Kit: Image 1 Expand Image
    Simple WesternTM analysis of lysates (0.1 mg/ml) from Jurkat cells using FoxO3a (D19A7) Rabbit mAb #12829. The virtual lane view (left) shows the target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the JessTM Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230kDa.
    Forkhead Signaling Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from 293T, MRK-nu-1 and Jurkat cells using FoxO3a (D19A7) Rabbit mAb (upper) and Akt (pan) (C67E7) Rabbit mAb #4691 (lower).
    Forkhead Signaling Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from 293T cells, untreated (-) or treated with LY294002 #9901 (50 μM, overnight; +) and Wortmannin #9951 (1 μM, overnight; +), using Phospho-FoxO3a (Ser253) (D18H8) Rabbit mAb (upper) or FoxO3a (D19A7) Rabbit mAb #12829 (lower).
    Forkhead Signaling Antibody Sampler Kit: Image 4 Expand Image
    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).
    Forkhead Signaling Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from 293T cells, either wild type (+/+) or FoxO1 (-/-), using FoxO1 (C29H4) Rabbit mAb (upper) and FoxO3a (75D8) Rabbit mAb #2497 (lower). 
    Forkhead Signaling Antibody Sampler Kit: Image 6 Expand Image
    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.
    Forkhead Signaling Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from COS cells, serum starved or serum treated, using Phospho-Fox01 (Ser256) Antibody.
    Forkhead Signaling Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from HT29 cells, serum starved or serum treated, using Phospho-Fox01 (Thr24)/Fox03a (Thr32) Antibody.
    Forkhead Signaling Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from serum starved and serum-treated NIH/3T3 cells as well as untreated and LY294002/Wortmannin-treated MDA-MB-468 cells, using Phospho-Fox03a (Ser318/321) Antibody (upper) or Akt Antibody #9272 (lower).
    Forkhead Signaling Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from Ramos and Jurkat cells, using Fox04 Antibody.
    Forkhead Signaling Antibody Sampler Kit: Image 11 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using FoxO3a (D19A7) Rabbit mAb.
    Forkhead Signaling Antibody Sampler Kit: Image 12 Expand Image
    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.
    Forkhead Signaling Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from IGROV-1 and COS-7 cells using FoxO1 (C29H4) Rabbit mAb.
    Forkhead Signaling Antibody Sampler Kit: Image 14 Expand Image
    Immunohistochemical analysis of paraffin-embedded PC-3 (upper) and MRK-nu-1 (lower) cell pellets, treated with Human Insulin-like Growth Factor I (hIGF-I) #8917 (left) or LY294002 #9901 (right), using FoxO3a (D19A7) Rabbit mAb.
    Forkhead Signaling Antibody Sampler Kit: Image 15 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using FoxO3a (D19A7) Rabbit mAb.
    Forkhead Signaling Antibody Sampler Kit: Image 16 Expand Image
    Immunoprecipitation of FoxO1 from C6 extracts. Lane 1 is 10% input, lane 2 is FoxO1 (C29H4) Rabbit mAb, and lane 3 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900. Western blot analysis was perfomed using FoxO1 (C29H4) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
    Forkhead Signaling Antibody Sampler Kit: Image 17 Expand Image
    Immunohistochemical analysis of paraffin-embedded metastatic SKOV3 tumor in mouse lung using FoxO3a (D19A7) Rabbit mAb. Note nuclear staining in adjacent normal lung.
    Forkhead Signaling Antibody Sampler Kit: Image 18 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon using FoxO1 (C29H4) Rabbit mAb.
    Forkhead Signaling Antibody Sampler Kit: Image 19 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using FoxO1 (C29H4) Rabbit mAb.
    Forkhead Signaling Antibody Sampler Kit: Image 20 Expand Image
    Confocal immunofluorescent analysis of PC-3 cells, treated with Human Insulin-like Growth Factor I (hIGF-I) #8917 (left) or LY294002 #9901 (right), using FoxO3a (D19A7) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
    Forkhead Signaling Antibody Sampler Kit: Image 21 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lymphoma using FoxO1 (C29H4) Rabbit mAb.
    Forkhead Signaling Antibody Sampler Kit: Image 22 Expand Image
    Flow cytometric analysis of MRK-nu-1 cells (blue) and Jurkat cells (green) using FoxO3a Rabbit mAb (solid line) compared to a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Forkhead Signaling Antibody Sampler Kit: Image 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded IGROV-1 cell pellets, LY294002-treated (left) or insulin-treated (right), using FoxO1 (C29H4) Rabbit mAb. Note the cytoplasmic localization of FoxO1 upon Akt activation.
    Forkhead Signaling Antibody Sampler Kit: Image 24 Expand Image
    Immunohistochemical analysis of paraffin-embedded SKOV-3 xenograft using FoxO1 (C29H4) Rabbit mAb.
    Forkhead Signaling Antibody Sampler Kit: Image 25 Expand Image
    Confocal immunofluorescent analysis of IGROV-1 cells, LY294002-treated (left) or insulin-treated (right), using FoxO1 (C29H4) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5 (fluorescent DNA dye).
    Forkhead Signaling Antibody Sampler Kit: Image 26 Expand Image
    Flow cytometric analysis of HL-60 cells (blue) and IGROV-1 cells (green) using FoxO1 (C29H4) Rabbit mAb (solid lines) or a 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.
    Forkhead Signaling Antibody Sampler Kit: Image 27 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from mouse liver tissue and either FoxO1 (C29H4) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Mouse Pdk4 Promoter Primers #31195, mouse Elk4 upstream primers and SimpleChIP® Mouse RPL30 Intron 2 Primers #7015. 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.