Phospho 4ebp1

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Phospho-4EBP1 is a laboratory product that detects the phosphorylation of the 4EBP1 protein. 4EBP1 is a regulator of protein synthesis and plays a role in the mTOR signaling pathway. The Phospho-4EBP1 product allows for the identification and quantification of the phosphorylated form of 4EBP1 in biological samples.

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Lab products found in correlation

Close spelling variants of this product

Anti-phospho-4EBP1 (57 citations) Rabbit anti-phospho-4EBP1 (15 citations) Phospho-4EBP1 T37/46 (10 citations) Anti-phospho-4EBP1 (Thr70) (7 citations) Anti-phospho Thr37/46 4EBP1 (2 citations) Phospho-4EBP1(T37 (2 citations) Anti-phospho-4EBP1 rabbit antibody (2 citations) Phospho (Ser65) 4EBP1 (2 citations)

120 protocols using phospho 4ebp1

Western Blot Analysis of 4EBP-1 and HIF-1α

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Whole cell protein extractions were performed as previously described [19 (link)]. Western blots analyses were performed with commercially available antibodies, 4EBP-1 (Cell signaling technology, #9644), HIF-1α (Abcam, ab51608) and Tubulin (Cell signaling technology, #2144S). Phospho-4EBP-1 (Thr37/46), non-phospho-4EBP-1 (Thr37/46), Phospho-4EBP-1 (Ser65) and Phospho-4EBP-1 (Thr70) were purchased as 4E-BP antibody sampler kit (Cell signaling technology, #9955). All experiments were repeated at least three times.

Chen L.Y., Wang L., Ren Y.X., Pang Z., Liu Y., Sun X.D., Tu J., Zhi Z., Qin Y., Sun L.N, & Li J.M. (2020). The circular RNA circ-ERBIN promotes growth and metastasis of colorectal cancer by miR-125a-5p and miR-138-5p/4EBP-1 mediated cap-independent HIF-1α translation. Molecular Cancer, 19, 164.

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Immunoblotting of Phosphorylated Proteins

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Tissue samples were collected and flash frozen. After 24 h, protein was extracted and immunoblotting performed as previously described.^{43 (link)} The membranes were blocked with 5% non-fat dry milk for 1 h and then probed with primary antibodies against phospho RPS6 (Ser235/236, 4858, Cell Signaling Technology), phospho AKT (Ser473, 4060, Cell Signaling Technology), phospho 4EBP1 (Thr37/46, 2855, Cell Signaling Technology), total RPS6 (2217, Cell Signaling Technology), total AKT (4691, Cell Signaling Technology) or total 4EBP1(9644, Cell Signaling Technology) in bovine serum albumin at 1:1000. Anti-GAPDH antibody (8884, Cell Signaling Technology) was used as a loading control at a ratio of 1:5000.

Payne S.N., Maher M.E., Tran N.H., Van De Hey D.R., Foley T.M., Yueh A.E., Leystra A.A., Pasch C.A., Jeffrey J.J., Clipson L., Matkowskyj K.A, & Deming D.A. (2015). PIK3CA mutations can initiate pancreatic tumorigenesis and are targetable with PI3K inhibitors. Oncogenesis, 4(10), e169-.

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Comprehensive Signaling Pathway Analysis

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PTEN(9559), p44/42(Erk1/2)(9107), phospho-p44/42(Thr202/Tyr204)(9101), p38MAPK(8690), phospho-p38MAPK(Thr180/Tyr182)(4511), eIF4E(9742), phospho-eIF4E(Ser209)(9741), Mnk(2195), phospho-Mnk(Thr197/202)(2111), phospho-4EBP1(Ser65)(9451), AKT(4685), phospho-Akt(Ser473)(9271), mTOR(2983), phospho-mTOR(Ser2448)(2971), phospho-p70S6K(Thr421/Ser424)(9205), AR(3202), α-Tubulin(2125), c-Myc(9402), Survivin(2808), Cyclin D1(2978), BCL-2(2872), p-eIF4G(2441) and EGFR(2232) were from Cell Signaling Technology (Beverly, MA), 4EBP1(6936), AR(N-20)(816), β-Actin(130656) were from Santa Cruz Biotechnology (Santa Cruz, CA). GAPDH(MAB374) was from EMD Millipore, Darmstadt, Germany. phospho-eIF4E(Ser209)(ab76256) for immunohistochemistry and immunofluorescence were from Abcam, Cambridge, UK. Ki67(7240) antibody was from DAKO via Agilent Technologies, Santa Clara, CA.

D’Abronzo L.S., Bose S., Crapuchettes M.E., Beggs R.E., Vinall R.L., Tepper C.G., Siddiqui S., Mudryj M., Melgoza F.U., Durbin-Johnson B.P., deVere White R.W, & Ghosh. P.M. (2017). The Androgen Receptor is a negative regulator of eIF4E Phosphorylation at S209: Implications for the use of mTOR inhibitors in advanced prostate cancer. Oncogene, 36(46), 6359-6373.

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Immunoblotting and Immunofluorescence Antibody Protocol

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Immunoblot analyses were performed using the following antibodies: Akt, Aktp^S473, p70S6K, phospho-p70S6K, 4EBP1, ERK1/2, Atg5, ULK, ERK1/2, phopho-ERK1/2, p38, phospho-p38, phospho-NFκB and phospho-4EBP1 were all purchased from Cell Signaling. FAK (Santa Cruz Biotechnology, Inc.), actin (Cytoskeleton), LC3 (Novus) and FAKp^Y397 (BD Transduction Laboratories) were purchased from the suppliers indicated. Monoclonal anti-HA (16B12) and polyclonal anti-HA (HA.11) were both purchased from Covance. For immunofluorescence studies, LC3 (Millipore), mTOR (Cell Signaling), LAMP-1 and LAMP-2 (Developmental Studies Hybridoma Bank, University of Iowa), p62 (abcam) and ubiquitin (FK2, Enzo Life Sciences) were from the suppliers indicated. Secondary antibodies included goat anti-mouse-Cy3, donkey anti-rabbit Alexa Fluor 488 and goat anti-rat Alexa Fluor 555 and were purchased from Invitrogen. For flow cytometry, TLR4 (Sa15-21; Akashi et al., 2003) was conjugated to biotin and was a kind gift from Jonathan Kagan (Harvard Medical School, Boston, MA). Anti- strepavidin-APC was purchased from Biolegend.

Owen K.A., Meyer C.B., Bouton A.H, & Casanova J.E. (2014). Activation of Focal Adhesion Kinase by Salmonella Suppresses Autophagy via an Akt/mTOR Signaling Pathway and Promotes Bacterial Survival in Macrophages. PLoS Pathogens, 10(6), e1004159.

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Western Blot Analysis of Protein Signaling

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Protein lysates were prepared in RIPA lysis buffer, and separated on 10% SDS–polyacrylamide gel electrophoresis gels by electrophoresis. Subsequently, the lysates were transferred to nitrocellulose membranes and probed with the following antibodies and concentrations: HIF-2α 1:1,000 (Novus #NB100-122), HIF-1α 1:1,000 (Cayman Chemicals #1006421), caspase-3 1:1,000 (Cell Signaling #9662 Danvers, MA, USA), GAPDH 1:2,000 (Cell Signaling #2118), β-tubulin 1:1,500 (Cell Signaling #2146), phospho-4E-BP1 1:1,000 (S65, Cell Signaling #9451), 4E-BP1 1:1,000 (Cell Signaling #9452), phospho-S6K1 1:1,000 (T389, Cell Signaling #9205), c-Myc 1:5,000 (Abcam #32072), S6K1 1:1,000 (Cell Signaling #2708), phospho-AKT 1:1,000 (S473, Cell Signaling #9271), AKT 1:1,000 (Cell Signaling #9272), phospho-EGFR 1:1,000 (Y1068, Cell Signaling #3777), EGFR 1:1,000 (Cell Signaling #4267) ANO1 1:500 (Abcam ab64085), phospho-CAMKIIα 1:1,000 (T286, Cell Signaling #12716) and CAMKII 1:1,000 (Cell Signaling #11945). Uncropped immunoblot images are included in Supplementary Fig. 8.

Nakazawa M.S., Eisinger-Mathason T.S., Sadri N., Ochocki J.D., Gade T.P., Amin R.K, & Simon M.C. (2016). Epigenetic re-expression of HIF-2α suppresses soft tissue sarcoma growth. Nature Communications, 7, 10539.

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Immunoblotting and Antibody Detection

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Immunoblotting was performed as described previously (Jacobs et al., 2008). Primary antibodies were followed by mouse- or rabbit-conjugated horseradish peroxidase (HRP). HRP-conjugated antibodies (anti-mouse or anti-rabbit IgG HRP conjugate, Promega) were detected by enhanced chemiluminescence detection (Thermofisher). This included the following antibodies: ACC (3662, Cell Signaling), phospho-ACC (3661, Cell Signaling), pan-AMPKα (2532, Cell Signaling), phospho-AMPKα (2535, Cell Signaling), 4EBP1 (9644, Cell Signaling), phospho-4EBP1 (2855, Cell Signaling), c-myc (9402, Cell Signaling), phospho-mTOR (5536, Cell Signaling), Activated Notch (ab8925, Abcam), RAPTOR (2280, Cell Signaling), phospho-RAPTOR (2083, Cell Signaling), S6 (2217, Cell Signaling), phospho-p70 S6K (9204, Cell Signaling), p70 S6K (2708, Cell Signaling), phosphor-TSC2 (5584, Cell Signaling), TSC2 (3612, Cell Signaling). Alternatively, primary antibodies were followed by fluorescently labeled anti-mouse or rabbit antibodies (LiCor) and imaged using the Odyssey infrared imaging system (LiCor). This included the following antibodies: Glut1 (ab652, Abcam), hexokinase 2 (2867, Cell Signaling), hexokinase 1 (ab104835, Abcam), cytochrome C (556433, BD Biosciences), β-actin (A5441, Sigma), phospho-S6 (4858, Cell Signaling). Western blots were quantified using ImageJ software.

Kishton R.J., Barnes C.E., Nichols A.G., Cohen S., Gerriets V.A., Siska P.J., Macintyre A.N., Goraksha-Hicks P., de Cubas A.A., Liu T., Warmoes M.O., Abel E.D., Yeoh A.E., Gershon T.R., Rathmell W.K., Richards K.L., Locasale J.W, & Rathmell J.C. (2016). AMPK is essential to balance glycolysis and mitochondrial metabolism to control T-ALL cell stress and survival. Cell metabolism, 23(4), 649-662.

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Comprehensive Antibody Panel for Cellular Analysis

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Antibodies to RKIP (CST#5291), AMPKα (CST#5832), phospho-AMPKα (T172) (CST#8208), p70S6K (CST#2708), phospho-p70S6K (CST#9208), 4EBP1 (CST#9644), phospho-4EBP1 (CST#9459), STAT3 (CST#9132), phospho-STAT3 (Tyr705) (CST#9131) and phospho-STAT3 (Ser727) (CST#9134) were purchased from Cell Signaling Technology. Antibodies to E-Cadherin (20874-1-AP) and vimentin (10366-1-AP) were obtained from Proteintech Group. Anti-ANXA7 antibody (sc-17815) used for co-immunoprecipitation and anti-p-Thr antibody (sc-5267) were obtained from Santa Cruz Biotechnology. Anti-ANXA7 antibody (A4475 MSDS) used for western blot and antibody to β-actin (A1978 MSDS) were purchased from Sigma. Horseradish peroxidase-conjugated secondary antibodies for Western blot were from Santa Cruze Biotechnology. Secondary antibody for immunofluorescence was donkey anti-rabbit IgG Alexa Fluor-488 (CA21206s, Invitrogen).

Liu S., Li X., Lin Z., Su L., Yan S., Zhao B, & Miao J. (2017). SEC-induced activation of ANXA7 GTPase suppresses prostate cancer metastasis. Cancer letters, 416, 11-23.

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Protein Expression Profiling in CLL Cells

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CLL cells were left untreated, treated with single agents, or treated with a combination of a Pim kinase inhibitor plus ABT-737 or ABT-199 as described above. The cell pellets were washed with ice-cold PBS and lysed at 4°C in radioimmunoprecipitation assay buffer supplemented with 1 mini Complete^™ Protease Inhibitor (Roche) tablet per 10 mL of buffer. The lysate protein content was measured using a DC protein assay kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Aliquots (30–50 μg) of total protein were loaded onto 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (GE Osmonics Labstore, Minnetonka, MN) as previously described [11 ]. The membranes were blocked at room temperature for 1 h in Odyssey blocking buffer (LI-COR Inc., Lincoln, NE) and then incubated overnight at 4°C with the following primary antibodies: Bcl-2 (Dako, Carpinteria, CA), Mcl-1, Bcl-X_L (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-4E-BP1 (Thr 37/46), total 4E-BP1, phospho-p70S6K (Thr 389), or GAPDH (Cell Signaling Technology, Danvers, MA), and PARP (BD Pharmingen). After washing, the membranes were incubated with infrared-labeled secondary antibodies (LI-COR, Lincoln, NE) for 1 h and visualized using a LI-COR Odyssey Infrared Imager.

Cervantes-Gomez F., Lavergne B., Keating M.J., Wierda W.G, & Gandhi V. (2015). Combination of Pim kinase inhibitors and Bcl-2 antagonists in chronic lymphocytic leukemia cells. Leukemia & lymphoma, 57(2), 436-444.

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Western Blot Assay of Signaling Pathways

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The Western blots were carried out according to the standard protocol. The following primary antibodies were used: phospho‐Akt (Thr308) rabbit mAb (13038), phospho‐Akt rabbit mAb (Ser 473) (4060), anti‐Akt (pan) rabbit mAb (4691), phospho‐P70s6k rabbit mAb (9205), P70s6k rabbit mAb (2708), phospho‐4ebp1 (#13396, monoclonal), anti‐4ebp1 (#9644), anti‐phospho‐B‐Raf (#2696, polyclonal), anti‐B‐Raf (#9433, monoclonal), anti‐phospho‐Erk (#4370, monoclonal), anti‐Erk (#4695, monoclonal) (all from Cell Signaling Technology, Beverly, MA, USA). The band density was quantified using ImageJ software (NIH, Rockville, MD, USA).

Chang F., Zhang Y., Mi J., Zhou Q., Bai F., Xu X., Fisher D.E., Sun Q, & Wu X. (2018). ROCK inhibitor enhances the growth and migration of BRAF‐mutant skin melanoma cells. Cancer Science, 109(11), 3428-3437.

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Western Blot Analysis of Protein Signaling

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Protein lysates were prepared in either RIPA or CHAPS buffer and quantified using the BCA Protein Assay (Thermo Scientific). Protein was separated on 4%–20% SDS-PAGE gels, transferred to PVDF membranes, and probed with antibodies against CPS1 (ab3682), β-actin (ab8227), ASS1 (clone 2B10, ab124465), cyclophilin B (clone EPR12703(B), ab178397), total AMPK (Cell Signaling, #2603), phospho-AMPK (Cell signaling, #2531), total ACC (Cell Signaling, #3662), phospho-ACC (Cell Signaling, #11818), LKB1 (Cell Signaling, #3050), γH2AX (Cell Signaling, #9718), total CAD (Cell Signaling, #11933), phospho-CAD (Cell Signaling, #12662), NOS3 (BD, 610298), phospho-S6 (Cell Signaling, #2211), phospho-4E-BP1 (Cell Signaling, #2855), CREB1 (Santa Cruz, sc-186X), FOXA1 (ab23738), TEAD4 (ab58310).

Kim J., Hu Z., Cai L., Li K., Choi E., Faubert B., Bezwada D., Rodriguez-Canales J., Villalobos P., Lin Y.F., Ni M., Huffman K.E., Girard L., Byers L.A., Unsal-Kacmaz K., Peña C.G., Heymach J.V., Wauters E., Vansteenkiste J., Castrillon D.H., Chen B.P., Wistuba I., Lambrechts D., Xu J., Minna J.D, & DeBerardinis R.J. (2017). CPS1 maintains pyrimidine pools and DNA synthesis in KRAS/LKB1-mutant lung cancer cells. Nature, 546(7656), 168-172.

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