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Pt37 46 4ebp1

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The PT37/46-4EBP1 is a phospho-specific antibody that recognizes the phosphorylated forms of 4EBP1 at Thr37/46. 4EBP1 is a member of the eukaryotic translation initiation factor 4E-binding protein (4E-BP) family, which regulates the activity of the translation initiation factor eIF4E.

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Lab products found in correlation

Pt389 s6k1, by Cell Signaling Technology (4 mentions) 4e bp1, by Cell Signaling Technology (4 mentions) Ps473 akt, by Cell Signaling Technology (2 mentions) Pt308 akt, by Cell Signaling Technology (2 mentions) Raptor, by Cell Signaling Technology (2 mentions) P s235 236 s6, by Cell Signaling Technology (2 mentions) P irs1 s1101, by Cell Signaling Technology (1 mentions) C ebpβ, by Santa Cruz Biotechnology (1 mentions) Irs 1, by Cell Signaling Technology (1 mentions) C ebpδ, by Santa Cruz Biotechnology (1 mentions) P ser235 236 s6, by Cell Signaling Technology (1 mentions) 9 10 3h n oleic acid, by PerkinElmer (1 mentions) Eif4e, by Cell Signaling Technology (1 mentions) P70s6k, by Cell Signaling Technology (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Pras40, by Cell Signaling Technology (1 mentions) Pt172 ampk, by Cell Signaling Technology (1 mentions) Cos 7 cells, by American Type Culture Collection (1 mentions) Nih3t3 cells, by American Type Culture Collection (1 mentions) Erk1 2, by Cell Signaling Technology (1 mentions)

4 protocols using pt37 46 4ebp1

1

Antibody Sources for Signaling Pathway Analysis

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Antibodies against the following proteins were obtained from the following sources: alpha-tubulin (ab11304) from Abcam; DEPTOR (NBP149674) from Novus Biologicals; raptor (for IP, A300-553A) from Bethyl laboratories; PLD2 (sc-25513), PPARγ (sc-7196), C/EBPα (sc-61) and aP2 (sc-271529) from Santa Cruz Biotechnology; C/EBPβ (#3087), C/EBPδ (#2318), IRS-1 (#2382), pS636/639-IRS-1 (#2388), pS1101-IRS-1 (#2385), mTOR (#2972), raptor (#2280), Akt (#9272), pS473-Akt (#4051), pT308-Akt (#9275), S6K1 (#9202), pT389-S6K1 (#9234), S6 (#2217), pSer235/236-S6 (#4856), 4EBP1 (#9644), and pT37/46-4EBP1 (#2855) from Cell Signaling Technology. The PLD1 antibody was provided by Dr. Jie Chen from the University of Illinois at Urbana-Champaign16 (link). All secondary antibodies were from Jackson ImmunoResearch Laboratories Inc. [9,10-3H(N)]-oleic acid was from PerkinElmer. C8-PA (830842C) was from Avanti. All other reagents were from Sigma-Aldrich.
Song H.I, & Yoon M.S. (2016). PLD1 regulates adipogenic differentiation through mTOR - IRS-1 phosphorylation at serine 636/639. Scientific Reports, 6, 36968.
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2

Immunoblotting Analysis of Cellular Signaling Proteins

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Total cellular protein was extracted and immunoblotted, using 20 μg of protein as described (22 (link),23 (link)). Antibodies from Cell Signaling Technology (Danvers, Massachusetts, USA) are; p70 S6K (Cat # 9202S), P-T389 S6K1 (Cat# 9205S), 4EBP1 (Cat# 9644P), P-T70 4EBP1 (Cat# 9455S), P-T37/46 4EBP1 (Cat# 2855L), eIF4E (Cat# 2067S), AMPK (Cat# 2793S), P-T172 AMPK (Cat# 2531S), mTOR (Cat# 2983S), P-2448 mTOR (Cat# 2971S), Raptor (Cat# 2280S), P-S792 Raptor (Cat# 2083S), TSC2 (Cat# 3990S), P-S1387 TSC2 (Cat# 5584S), PRAS40 (Cat# 2610S), P-T246 PRAS40 (Cat# 2640S), and β-Actin (Cat# 3700P). p53 antibody (Cat# OP43) is from Calbiochem (EMD, Darmstadt, Germany). Sestrin2 antibody was (Cat# 10795-1-AP) from Proteintech Group Inc. (Chicago, USA). Chemiluminescence was detected using SuperSignal Substrate (Thermo Scientific, Rockford, IL, USA.).
Agarwal S., Bell C.M., Taylor S.M, & Moran R.G. (2015). p53 Deletion or Hot-spot Mutations Enhance mTORC1 Activity by Altering Lysosomal Dynamics of TSC2 and Rheb. Molecular cancer research : MCR, 14(1), 66-77.
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3

Western Blot Analysis of Signaling Pathways

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Cells were rinsed once with ice-cold PBS and lysed in ice-cold lysis buffer (50 mM HEPES [pH 7.4], 40mM NaCl, 2mM EDTA, 1.5mM sodium orthovanadate, 50mM NaF, 10mM pyrophosphate, 10mM glycerophosphate, and 1% Triton X-100, and one tablet of EDTA-free complete protease inhibitors [Roche] per 25ml). Cell lysates were cleared by centrifugation at 13,000rpm for 10 min. Proteins extracts were denatured by the addition of sample buffer, boiled for 5min, resolved by SDS-PAGE, and analyzed by immunoblotting. Western blot analyses were performed according to standard procedures. Antibodies from Cell Signaling were used for detection of P-T389-S6K1 (#9234), S6K1 (#2708), P-S235/236-S6 (#2211), S6 (#2217), P-T37/46-4EBP1 (#2855), 4EBP1 (#9644), P-T308-AKT (#2965), P-S743-AKT (#4060), AKT (#4691). For detection of β-actin we used an antibody from Sigma (#A5441).
Ortega-Molina A., Deleyto-Seldas N., Carreras J., Sanz A., Lebrero-Fernández C., Menéndez C., Vandenberg A., Fernández-Ruiz B., Marín-Arraiza L., de la Calle Arregui C., Belén Plata-Gómez A., Caleiras E., de Martino A., Martínez-Martín N., Troulé K., Piñeiro-Yáñez E., Nakamura N., Araf S., Victora G.D., Okosun J., Fitzgibbon J, & Efeyan A. (2019). Oncogenic Rag GTPase signaling enhances B cell activation and drives follicular lymphoma sensitive to pharmacological inhibition of mTOR. Nature metabolism, 1(8), 775-789.
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4

Investigating mTOR Signaling Pathway

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NIH3T3 cells and COS-7 cells were purchased from American Type Culture Collection (ATCC) and used within six months after cells were ordered. These cell lines were not authenticated in our laboratory. Rapamycin, 5-Bromo-2′-deoxyuridine (BrdU) and cycloheximide were obtained from Sigma. PF-4708671 was purchased from Selleckchem. MG132 was purchased from CalBiochem. Antibodies directed against Myc-tag (9E10), Flag-tag, and HA-tag were obtained from Covance. Antibodies directed against VPS34 (cat#4263), VPS15 (cat#14580), Beclin 1 (cat#3495), pT389-S6K1 (cat#9234), S6K1 (cat#2708), pT37/46-4EBP1 (cat#2855), 4EBP1 (cat#9644), pS235/236-S6 (cat#4858), S6 (cat#2217), pErk1/2 (cat#4370), Erk1/2 (cat#4695), pS473-Akt(cat#4060), Akt (cat#4685), RheB (cat#13879), TSC1 (cat#6935), TSC2 (cat#4308), BrdU (cat#5292), and Cyclin E (cat#4129) were purchased from Cell Signaling Technology. Fluorescently tagged antibodies were obtained from Invitrogen. The class III PI3K lipid kinase ELISA kit was purchased from Echelon Biosciences. The RheB activation kit was purchased from Abcam. 4-6 weeks old SCID mice were ordered from National Cancer Institute (NCI), NIH (Bethesda, MD).
Mohan N., Shen Y., Dokmanovic M., Endo Y., Hirsch D.S, & Wu W.J. (2016). VPS34 regulates TSC1/TSC2 heterodimer to mediate RheB and mTORC1/S6K1 activation and cellular transformation. Oncotarget, 7(32), 52239-52254.
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