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9946
Forkhead Signaling Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Forkhead Signaling Antibody Sampler Kit #9946

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Forkhead Signaling Antibody Sampler Kit: Image 1

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 2

Western blot analysis of extracts from COS cells, serum starved or serum treated, using Phospho-Fox01 (Ser256) Antibody.

Forkhead Signaling Antibody Sampler Kit: Image 3

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 4

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.

Forkhead Signaling Antibody Sampler Kit: Image 5

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 6

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 7

Western blot analysis of extracts from Ramos and Jurkat cells, using Fox04 Antibody.

Forkhead Signaling Antibody Sampler Kit: Image 8

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 9

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 10

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 11

Flow cytometric analysis of IGROV-1 (green) and Jurkat (blue) using FoxO1 (C29H4) Rabbit mAb. Anti-rabbit (H+L), F(ab')2 Fragment (PE Conjugate) #8885 was used as a secondary antibody.

Forkhead Signaling Antibody Sampler Kit: Image 12

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 13

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 14

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using FoxO3a (D19A7) Rabbit mAb.

Forkhead Signaling Antibody Sampler Kit: Image 15

Immunohistochemical analysis of paraffin-embedded human colon using FoxO1 (C29H4) Rabbit mAb.

Forkhead Signaling Antibody Sampler Kit: Image 16

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 17

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using FoxO1 (C29H4) Rabbit mAb.

Forkhead Signaling Antibody Sampler Kit: Image 18

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using FoxO3a (D19A7) Rabbit mAb.

Forkhead Signaling Antibody Sampler Kit: Image 19

Immunohistochemical analysis of paraffin-embedded human lymphoma using FoxO1 (C29H4) Rabbit mAb.

Forkhead Signaling Antibody Sampler Kit: Image 20

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 21

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 22

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 23

Immunohistochemical analysis of paraffin-embedded SKOV-3 xenograft using FoxO1 (C29H4) Rabbit mAb.

Forkhead Signaling Antibody Sampler Kit: Image 24

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 25

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 26

Western blot analysis of extracts from IGROV-1 and COS-7 cells using FoxO1 (C29H4) Rabbit mAb.

To Purchase # 9946T
Product # Size Price
9946T
1 Kit  (8 x 20 µl) $ 554

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-FoxO1 (Thr24)/FoxO3a (Thr32) Antibody 9464 20 µl
  • WB
  • IP
H M R Mk 78 to 82, 95 Rabbit 
Phospho-FoxO1 (Ser256) Antibody 9461 20 µl
  • WB
H M R Mk 82 Rabbit 
Phospho-FoxO1 (Thr24)/FoxO3a (Thr32)/FoxO4 (Thr28) (4G6) Rabbit mAb 2599 20 µl
  • WB
H M Mk 65, 78 to 82, 95 Rabbit 
FoxO1 (C29H4) Rabbit mAb 2880 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H M R Mk 78 to 82 Rabbit IgG
Phospho-FoxO3a (Ser253) (D18H8) Rabbit mAb 13129 20 µl
  • WB
H M R Mk 97 Rabbit IgG
Phospho-FoxO3a (Ser318/321) Antibody 9465 20 µl
  • WB
  • IP
H M R Mk 97 Rabbit 
FoxO4 Antibody 9472 20 µl
  • WB
H M R Mk 65 Rabbit 
FoxO3a (D19A7) Rabbit mAb 12829 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R 82 to 97 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product 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.

Specificity / Sensitivity

Phospho-FoxO1 (Thr24)/FoxO3a (Thr32) Antibody detects endogenous levels of FoxO1/FoxO3a only when phosphorylated at Thr24 of FoxO1 or Thr32 of FoxO3a. The antibody cross-reacts with phosphorylated FoxO4 at Thr28, but not with FoxO1 family members phosphorylated at other sites. Phospho-FoxO1 (Ser256) Antibody detects endogenous levels of FoxO1 only when phosphorylated at Ser256. The antibody cross-reacts with Fox04 phosphorylated at Ser193. 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. FoxO1 (C29H4) Rabbit mAb detects endogenous levels of total FoxO1 protein. The antibody does not detect exogenously expressed family members FoxO3a or FoxO4. Phospho-FoxO3a (Ser253) (D18H8) Rabbit mAb detects endogenous levels of FoxO3a only when phosphorylated at Ser253. This antibody may cross-react with FoxO1 when overexpressed and phosphorylated at Ser251 or FoxO4 when overexpressed and phosphorylated at Ser197. Phospho-FoxO3a (Ser318/321) Antibody detects endogenous levels of FoxO3a only when phosphorylated at Ser318/321. The antibody is expected to cross-react with FoxO1 when phosphorylated at Ser322/325 based on the peptide sequence. FoxO3a (D19A7) Rabbit mAb recognizes endogenous levels of total FoxO3a protein. The FoxO4 Antibody detects endogenous levels of FoxO4. The antibody is sensitive to phosphorylation within the antigen and preferrentially detects unphosphorylated FoxO4.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr28 of human Fox04, with a synthetic phosphopeptide corresponding to residues around Ser256 of human Fox01, or with a synthetic peptide corresponding to residues of human Fox04. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with recombinant protein specific to the carboxy terminus of human FoxO3 protein, with a GST-fusion protein corresponding to carboxy-terminal residues of human FoxO1, with a synthetic phosphopeptide corresponding to the sequence of human Fox03a, or with a synthetic phosphopeptide corresponding to residues surrounding Thr28 of human Fox04.

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.

Limited Uses

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For Research Use Only. Not For Use In Diagnostic Procedures.
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