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9955
4E-BP Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit
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4E-BP Antibody Sampler Kit #9955

Citations (8)
Immunoprecipitation of Phospho-4E-BP1 (Ser65) from MCF-7 cell extracts. 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 treated with 100 nM insulin for 30 minutes. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is Phospho-4E-BP1 (Ser65) Antibody. Western blot was performed using Phospho-4E-BP1 (Ser65) Antibody #9451.
Western blot analysis of extracts from A673 cells, untreated or nocodazole-treated (100 ng/ml, 16 hrs), using 4E-BP2 Antibody (upper) or 4E-BP1 Antibody #9452 (lower). Extracts were treated with lambda phosphatase NEB#P0753 (10,000 U/ml for 1 hour) to dephosphorylate both proteins.
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.
Western blot analysis of extracts from COS cells, treated with λ phosphatase or 20% FBS as indicated, using Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb (upper), Phospho-4E-BP1 (Thr37/46) Antibody #9459 (middle), and 4E-BP1 Antibody #9452 (lower).
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 blot analysis of extracts from 293 cells using 4E-BP1 Antibody #9644 (lower) and Phospho-4E-BP1 (Ser65) Antibody #9451 (upper). 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.
Western blot analysis of extracts from 293 cells, untreated or treated with 20% FBS for the indicated number of minutes, using Phospho-4E-BP1 (Thr70) Antibody (upper) or 4E-BP1 Antibody #9452 (lower).
Western blot analysis of extracts from control HeLa cells (Lane 1) or HeLa cells with a targeted mutation in the gene encoding 4E-BP1 (Lane 2) using 4E-BP1 (53H11) Rabbit mAb (upper) or β-actin (13E5) Rabbit mAb #4970 (lower). The change in 4E-BP1 molecular weight in the mutated HeLa cells confirms the specificity of the antibody for 4E-BP1.
Western blot analysis of bacterially expressed GST-4E-BP1 and of extracts from NIH/3T3 cells, using 4E-BP2 Antibody and 4E-BP1 Antibody #9452.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb.
Confocal immunofluorescent analysis of 293 shRNA Scramble cells either serum starved (far left); serum starved and treated with U0126 #9903 (10 μM, 2 hr), LY294002 #9901 (50 μM, 2 hr), and Rapamycin #9904 (10 nM, 2 hr) (center, left); serum starved and treated with l phosphatase (10,000 U/mL, 2 hr) ) (center, right); and 293 shRNA 4E-BP1/2 KO treated with U0126 #9903 (10 μM, 2 hr), LY294002 #9901 (50 μM, 2 hr), and Rapamycin #9904 (10 nM, 2 hr) (far right), using Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb (green). Red = Propidium Iodide (PI)/RNase Staining Solution.
Western blot analysis of extracts from various cell lines using 4E-BP1 (53H11) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, showing cytoplasmic and nuclear localization, using 4E-BP2 Antibody.
Immunohistochemical analysis of paraffin-embedded human lymphoma using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb.
Flow cytometric analysis of COS cells, untreated (blue) or λ phosphatase-treated (green), using Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb compared to a nonspecific negative control antibody (red).
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using 4E-BP1 (53H11) Rabbit mAb in the presence of control peptide (left) or 4E-BP1 blocking peptide #1053 (right).
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using 4E-BP2 Antibody.
Immunohistochemical analysis of paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right), using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb on SignalSlide (TM) Phospho-Akt (Ser473) IHC Controls #8101.
Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma using 4E-BP1 (53H11) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human follicular carcinoma (thyroid), using 4E-BP2 Antibody.
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).
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using 4E-BP1 (53H11) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded mouse spleen, 4E-BP1/2 wild type (left) or 4E-BP1 knockout (right), using 4E-BP1 (53H11) Rabbit mAb. 4E-BP wild type and knockout tissues kindly provided by Dr. Nahum Sonenberg, McGill University.
Confocal immunofluorescent analysis of HeLa cells using 4E-BP1 (53H11) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
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).
Confocal immunofluorescent analysis of 293 cells, expressing either nontargeting shRNA (left) or shRNA targeting 4E-BP1/2 (right), using 4E-BP1 (53H11) Rabbit mAb #9644 (green). Red = Propidium Iodide (PI)/RNase Staining Solution #4084.
Flow cytometric analysis of 293 cells, transfected with control shRNA (green) or 4E-BP1-specific shRNA (blue) using 4E-BP1 (53H11) 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.
Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, Wortmannin #9951, and U0126 #9903 (50 μM, 1 μM, and 10 μM, 2 hr; blue) using Phospho-4E-BP1 (Thr36/46) (236B4) 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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To Purchase # 9955
Cat. # Size Qty. Price Inventory
9955T		 1 Kit  (6 x 20 microliters)

Custom Formulation

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
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
Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb 4923 20 µl
  • WB
  • IF
  • F
 H M R Mk 15-20 Rabbit IgG
Phospho-4E-BP1 (Ser65) Antibody 9451 20 µl
  • WB
  • IP
 H M R Mk 15 to 20 Rabbit 
4E-BP1 (53H11) Rabbit mAb 9644 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
 H M R Mk 15-20 Rabbit IgG
4E-BP2 Antibody 2845 20 µl
  • WB
  • IP
  • IHC
 H M R Mk B 15 to 20 Rabbit 
Phospho-4E-BP1 (Thr70) Antibody 9455 20 µl
  • WB
  • IP
 H M R Mk 15 to 20 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
 Rab Goat 

Product Description

The 4E-BP Antibody Sampler Kit provides an economical means to investigate regulation of cap-dependent translation within the cell. The kit contains primary and secondary antibodies to perform two Western blots with each antibody.

Specificity / Sensitivity

Phospho-4E-BP1 (Thr37/46) Rabbit mAb detects endogenous levels of 4E-BP1 only when phosphorylated at Thr37 and/or Thr46, and may cross-react with 4E-BP2 and 4E-BP3 when phosphorylated at equivalent sites. Nonphospho-4E-BP1 (Thr46) (87D12) Rabbit mAb detects endogenous levels of 4E-BP1 only when dephosphorylated at Thr46. This antibody cross-reacts with 4E-BP2 and 4E-BP3 dephosphorylated at equivalent sites. Phospho-4E-BP1 (Ser65) Antibody detects endogenous levels of 4E-BP1 when phosphorylated at Ser65, and may also recognize 4E-BP1 when phosphorylated at Ser101. Phospho-4E-BP1 (Ser65) (174A9) Rabbit mAb detects endogenous levels of 4E-BP1 when phosphorylated at Ser65. Phospho-4E-BP1 (Thr70) Antibody detects endogenous levels of 4E-BP1 only when phosphorylated at Thr70. 4E-BP1 (53H11) Rabbit mAb detects endogenous levels of total 4E-BP1 protein. 4E-BP2 Antibody detects endogenous levels of total 4E-BP2 independent of phosphorylation and does not cross-react significantly with 4E-BP1.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr37 and Thr46 of mouse 4E-BP1, residues surrounding Thr46 of human 4E-BP1, or Ser112 of human 4E-BP1. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the residues at the carboxy-terminus of human 4E-BP2 (#2845), or phosphopeptides surrounding mouse Ser65 (#9451) and human Thr70 (#5078) 4E-BP1. Polyclonal antibodies were purified by protein A and peptide affinity chromatography.

Background

Translation repressor protein 4E-BP1 (also known as PHAS-1) inhibits cap-dependent translation by binding to the translation initiation factor eIF4E. Hyperphosphorylation of 4E-BP1 disrupts this interaction and results in activation of cap-dependent translation (1). Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase regulate 4E-BP1 activity (2,3). Multiple 4E-BP1 residues are phosphorylated in vivo (4). While phosphorylation by FRAP/mTOR at Thr37 and Thr46 does not prevent the binding of 4E-BP1 to eIF4E, it is thought to prime 4E-BP1 for subsequent phosphorylation at Ser65 and Thr70 (5).
4E-BP2 and 4E-BP3 share high sequence homology with 4E-BP1, including conservation of the major FRAP/mTOR-dependent phosphorylation sites. Preliminary data suggests that phosphorylation of 4E-BP2 is regulated in a similar manner to that of 4E-BP1, although phosphorylation of this protein has not been as extensively studied (6).

Pathways

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Limited Uses

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