Anti 4e bp1 antibody

Manufactured by Cell Signaling Technology

Sourced in United States

The Anti-4E-BP1 antibody is a laboratory reagent used in research applications. It is a highly specific antibody that recognizes the 4E-BP1 protein, a key regulator of protein synthesis. This antibody can be used to detect and quantify the expression levels of 4E-BP1 in various cellular and biological samples.

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6 protocols using anti 4e bp1 antibody

Platelet-activating Factor Signaling

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We purchased the following reagents: Platelet-activating factor (Avanti Polar Lipids, Alabaster, AL); Whole Blood anti-human CD15 microbeads (Miltenyi Biotec, Auburn, CA); Media-199 (Lonza Biologics, Alpharetta, GA); TOPRO-3 (Molecular Probes, Eugene, OR); Rapamycin (Calbiochem, Burlington, MA). We purchased the following antibodies from Cell Signaling Technology (Danvers, MA): anti-mTOR antibody (#2972), anti-phospho mTOR antibody (#2971), anti-4E-BP1 antibody (#9452), anti-phospho 4E-BP1 T37/46 antibody (#9459), anti-phospho 4E-BP1 Ser65 antibody (#9451), anti-phospho 4E-BP1 Thr70 antibody (#9455), anti-β-actin antibody. Anti-human PAF-receptor and anti-human IgG isotype-matched control antibodies were purchased from Abcam (Cambridge, MA). The goat-anti-rabbit Alexa Fluor 488 secondary antibody was purchased from Life Technologies (Carlsbad, CA) and the anti-IL6Rα primary antibody from Santa Cruz Biotechnology (Dallas, TX).

Campbell R.A., Cody M.J., Manne B.K., Zimmerman G.A, & Yost C.C. (2019). Interleukin 6 receptor alpha expression in PMNs isolated from prematurely born neonates: Decreased expression is associated with differential mTOR signaling.. Pediatric research, 86(1), 55-62.

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Protein Expression Analysis in Cells

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Cells were lysed using RIPA protein extraction reagent (Beyotime, China) supplemented with a protease inhibitor cocktail (Roche, USA). Western blotting was carried out as previously reported 17 (link). The proteins were detected using anti-LC3B antibody (Cell Signaling, USA), anti-4EBP1 antibody (Cell Signaling, USA), anti-mTOR antibody (Cell Signaling, USA), anti-p-mTOR antibody (Cell Signaling, USA), anti-Akt antibody (Diagbio, China), anti-p-Akt antibody (Diagbio, China), anti-Erk antibody (Diagbio, China), anti-p-Erk antibody (Diagbio, China), anti-GAPDH antibody (Sigma-Aldrich, USA) and corresponding secondary antibodies.

Wang B., Deng Y., Jin J., Wu Y, & Shen L. (2021). Long Noncoding RNA LIT3527 Knockdown induces Apoptosis and Autophagy through inhibiting mTOR pathway in Gastric Cancer Cells. Journal of Cancer, 12(16), 4901-4911.

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Platelet-activating Factor Signaling

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Immunohistochemical Analysis of 4E-BP1 in Colorectal Tissues

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Non-cancerous and colorectal cancer tissues were sectioned from patients and formalin-fixed and paraffin-embedded. These sections were deparaffinized with xylen, rehydrated in different concentration of alcohol and immersed in 3% H₂0₂ for removing endogenous peroxidase. Sections were heated in a microwave in citrate buffer (pH 6.0) for antigen retrieval. Washing with PBS, the sections were blocked with 3% BSA/PBS for 30 min and incubated with anti-4E-BP1 antibody (Cell Signaling) at 4°C overnight. The streptavidin peroxidase method was performed (Dako, LSAB kit) to develop the signal.

Chao M.W., Wang L.T., Lai C.Y., Yang X.M., Cheng Y.W., Lee K.H., Pan S.L, & Teng C.M. (2015). eIF4E binding protein 1 expression is associated with clinical survival outcomes in colorectal cancer. Oncotarget, 6(27), 24092-24104.

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Signaling Pathway Analysis of LPA-Induced Autophagy

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Lysophosphatidic acid (LPA) (oleoyl C: 18:1) was obtained from Avanti Polar Lipids (Alabaster, AL, United States). 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonylamino)-3-methyl-5-isoxazolyl]benzylsulfanyl)propanoic acid (Kil6425) was purchased from Sigma (St. Louis, MO, United States). PI3K inhibitor LY294002, rapamycin, anti-phosphorylated mTOR (ser2448) antibody, anti-total mTOR antibody, anti-p70s6 kinase (Thr389) antibody, anti-phosphorylated p70s6 kinase, anti-4E-BP1 antibody, anti-phosphorylated 4E-BP1(Thr37/46) antibody, and anti-phosphorylated AMPKα (Thr172) antibody were obtained from Cell Signaling Technology (Beverly, MA, United States). Anti-p62/SQSTM1 (sequestosome1) polyclonal antibody and anti-beclin1 polyclonal antibody were from Proteintech Technology (Proteintech Group, Wuhan, China), and anti-LC3B polyclonal antibody and anti-GAPDH monoclonal antibody were obtained from Sigma (St. Louis, MO, United States). Lipofectamine^TM RNAiMAX, stealth siRNA, and siRNA negative control were purchased from life Technologies (Invitrogen, Carlsbad, CA, United States). The PowerUp^TM SYBR Green Master Mix assay (Applied Biosystems, Life Technologies, Foster City, CA, United States).

Yang J., Xu J., Han X., Wang H., Zhang Y., Dong J., Deng Y, & Wang J. (2018). Lysophosphatidic Acid Is Associated With Cardiac Dysfunction and Hypertrophy by Suppressing Autophagy via the LPA3/AKT/mTOR Pathway. Frontiers in Physiology, 9, 1315.

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Western Blot Antibody Characterization

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EGFP was detected by anti-GFP rabbit serum A-6455 (Life Technologies), ACTINB was detected by monoclonal anti-β-actin antibody (A5441, Sigma Aldrich), TUBA1A was detected by anti-alpha Tubulin antibody (ab80779, abcam), GAPDH was detected by Anti-GAPDH antibody (G4595, SIGMA Aldrich),PKR was detected by anti-PKR antibody (ab32052, abcam), p-PKR was detected by anti-PKR phospho T451 antibody (ab81303, abcam), eIF2-alpha was detected by anti EIF2S1 antibody (ab26197, abcam), p-eIF2-alpha was detected by anti-EIF2S1 phospho S51 antibody (ab32157, abcam), p-4E-BP1 was detected by anti-phospho-4E-BP1 (ser65) antibody (#9451, Cell Signaling Techonology) and 4E-BP1 was detected by anti-4E-BP1 antibody (#9452, Cell Signaling Technology). Secondary antibodies were horseradish peroxidase (HRP) conjugated Polyclonal Goat Anti-Rabbit antibody (P0448; Dako) and horseradish peroxidase (HRP) conjugated Polyclonal Goat Anti-Mouse antibody (P0447; Dako).

Takahashi H., Kozhuharova A., Sharma H., Hirose M., Ohyama T., Fasolo F., Yamazaki T., Cotella D., Santoro C., Zucchelli S., Gustincich S, & Carninci P. (2018). Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation. PLoS ONE, 13(2), e0183229.

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