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P4ebp

Manufactured by Cell Signaling Technology
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P4EBP is a laboratory reagent used to detect and quantify the phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). 4E-BP1 is a key regulator of protein synthesis and cell growth. The P4EBP product allows researchers to study the activation of the mTOR signaling pathway, which plays a crucial role in cellular processes such as cell proliferation, survival, and metabolism.

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Lab products found in correlation

P akt, by Cell Signaling Technology (3 mentions) P ampk, by Cell Signaling Technology (2 mentions) P p70s6k, by Cell Signaling Technology (2 mentions) P70s6k, by Cell Signaling Technology (2 mentions) Bcl 2, by Cell Signaling Technology (2 mentions) P s6k, by Cell Signaling Technology (2 mentions) P stat3, by Cell Signaling Technology (1 mentions) P raptor, by Cell Signaling Technology (1 mentions) Pras40, by Cell Signaling Technology (1 mentions) Lamin b1 d4q4z, by Cell Signaling Technology (1 mentions) P s6k1, by Cell Signaling Technology (1 mentions) C myc 9e10, by Santa Cruz Biotechnology (1 mentions) Anti raptor 24c12, by Cell Signaling Technology (1 mentions) Anti traf6 h274, by Santa Cruz Biotechnology (1 mentions) Traf6 h 274, by Santa Cruz Biotechnology (1 mentions) Anti bcl6 antibody, by BD (1 mentions) P pras40, by Cell Signaling Technology (1 mentions) Anti ha 3f10, by Roche (1 mentions) Protein assay kit, by Bio-Rad (1 mentions) Chemidoc, by Bio-Rad (1 mentions)

Close spelling variants of this product

Anti-p4EBP (2 citations) α -p4EBP (2 citations)

12 protocols using p4ebp

1

Isolation and Analysis of Cellular Fractions

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Nuclear and cytoplasmic extracts from naive T cells cultured for 3 days were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce), as previously described21 (link). The purity of the nuclear and cytoplasmic fractions was verified by probing with antibodies against Lamin B1 (D4Q4Z; 1,000×; Cell Signaling Technology). Whole-cell extracts were immunoprecipitated with an anti-Raptor (24C12; 100×; Cell Signaling Technology), anti-Def6 (Rabbit polyclonal; 100×21 (link)), anti-p62 (H-290; 50×; Santa Cruz), anti-TRAF6 (H274; 50×; Santa Cruz), or anti-HA (3F10; 50×; Roche Applied Science) antibodies. Antibodies to p-STAT3 (Y705; 1,000×), p-4E-BP (T37/46; 1,000×), 4E-BP (1,000×), p-S6K1 (S371; 1,000×), S6K1 (1,000×), p-AKT (S473; 1,000×), AKT (1,000×), p-AKT (T308; 1,000×), p-PRAS40 (T246; 1,000×), PRAS-40 (1,000×), p-AMPK (T172; 1,000×), AMPK (1,000×), p-Raptor (S792; 1,000×) and p62 (5114; 1,000×) were obtained from Cell Signaling Technology. Antibodies to IRF4 (M-17; 1,000×), TRAF6 (H274; 500×), and c-Myc (9E10; 500×) were obtained from Santa Cruz. Anti-Bcl6 antibody was obtained from BD (K112-91; 1,000×). Anti-Flag monoclonal antibody M2 (horseradish peroxidase (HRP)) was obtained from Sigma (1,000×).
Yi W., Gupta S., Ricker E., Manni M., Jessberger R., Chinenov Y., Molina H, & Pernis A.B. (2017). The mTORC1-4E-BP-eIF4E axis controls de novo Bcl6 protein synthesis in T cells and systemic autoimmunity. Nature Communications, 8, 254.
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2

Western Blot Assay for Protein Analysis

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Western blot assays were performed using the following antibodies: MCL-1 MoAb used at a dilution of 1:1000 (4572 Cell Signaling), vinculin MoAb (Sigma-Aldrich, dilution of 1:10,000), C-MET (Santa Cruz, 1:1000), P-MET (Cell Signaling, 1:1000), 4EBP (Cell Signaling, 1:1000), P-4EBP (Cell Signalling, 1:1000), S6 (Cell Signaling, 1:1000) and P-S6 (Cell Signaling, 1:1000). Antibodies were purchased from the indicated sources.
Cells were seeded (1.5 × 106 in adhesion and 4 × 106 in suspension) and resuspended after drug treatment in a urea buffer; then protein concentration was determined by a protein assay kit (Bio-Rad). Proteins (80 µg) were separated in 12% SDS–polyacrylamide gels, transferred onto a nitrocellulose membrane and probed against MCL-1 MoAb. The secondary antibody was mouse IgGκ-binding protein HRP. Proteins were visualized with Clarity Western ECL and analyzed with Chemidoc (Bio-Rad).
Cella C.A., Cazzoli R., Fazio N., De Petro G., Gaudenzi G., Carra S., Romanenghi M., Spada F., Grossi I., Pallavicini I., Minucci S, & Vitale G. (2023). Cabozantinib in neuroendocrine tumors: tackling drug activity and resistance mechanisms. Endocrine-Related Cancer, 30(12), e230232.
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3

Fly Intestine Immunofluorescence Staining

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Female fly intestines were dissected in PBS 1X solution and then fixed in Fixation Buffer containing 100 mM glutamic acid, 25 mM KCl, 20 mM MgSO4, 4 mM sodium phosphate, 1 mM MgCl2, and 4% formaldehyde for 45 minutes at room temperature. Samples were blocked using Blocking buffer containing PBS 1X, 0.5% BSA, and 0.1% Triton X-100. Samples were then incubated in the same buffer containing primary antibodies over-night at 4°C [anti-phospho-Histone H3 from Millipore (1:2000), anti-Beta-Galactosidase from DHSB (1:500), anti-Prospero from DHSB (1:200)]. Fluorescent secondary antibodies were obtained from Jackson Immunoresearch. DNA was stained using Hoechst and Visceral muscle was stained using Alexa Fluor 647 Phalloidin (from Invitrogen, 1:400). For Delta (from DHSB, 1:500), Sox21a (generated in the lab [43 (link)], 1:5000), p4EBP (from Cell signaling technologies, 1:200), zfh2 (kindly provided by Chris Doe, 1:200) and PDM1 (kindly provided by Xiaohang Yang and Cai Yu, 1:1000) staining samples were fixed in Fixation Buffer and Heptane, dehydrated with 100% Methanol and progressively re-hydrated in blocking Buffer. Confocal imaging was done using the Leica SP5 system and processed with Adobe Photoshop CS6.
Rojas Villa S.E., Meng F.W, & Biteau B. (2019). zfh2 controls progenitor cell activation and differentiation in the adult Drosophila intestinal absorptive lineage. PLoS Genetics, 15(12), e1008553.
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4

Western Blot Analysis of Cell Signaling

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For Western blotting, equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1 h with nonfat dry milk solution (5% in Tris-buffered saline) containing 0.1% Tween 20. The blots were probed with primary antibodies for p-p70S6K (catalog number 9208; Cell Signaling Technology), p70S6K (catalog number 9202; Cell Signaling Technology), p-4EBP (catalog number 2855; Cell Signaling Technology), 4EBP (catalog number 9644; Cell Signaling Technology), CAR (catalog number sc-15405; Santa Cruz Biotechnology), LC3 (catalog number L7543; Sigma), Bcl-2 (catalog number 2870; Cell Signaling Technology), and vp1 (catalog number M706401; Dako). Horseradish peroxidase (HRP)-conjugated anti-rabbit (catalog number 7074; Cell Signaling Technology) or anti-mouse IgG (catalog number 7076; Cell Signaling Technology) was used as a secondary antibody. Immunoreactive bands were visualized by enhanced chemiluminescence (Pierce).
Zhou L., He X., Gao B, & Xiong S. (2015). Inhibition of Histone Deacetylase Activity Aggravates Coxsackievirus B3-Induced Myocarditis by Promoting Viral Replication and Myocardial Apoptosis. Journal of Virology, 89(20), 10512-10523.
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5

Bufalin-Induced Cell Death Mechanisms

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Bufalin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethylsulfoxide (DMSO) at 0.4 mM and diluted with fresh medium to achieve the desired concentration. Caspase-3 colorimetric assay kit was purchased from BioVision, Inc. (U.S.A). 3-Methyladenine (3-MA), 3-(4,5-dimetrylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS), temozolomide (TM), Dihydroethidium (DHE), wortmannin (WORT), Chloroquine (CQ), monodancylcadaverin (MDC), acridine orange (AO) and tauroursodeoxycholate (TUDC) were purchased from Sigma (St. Louis, MO, USA). Rabbit antibodies specific for Cleaved caspase-3, Cleaved caspase-4, Cleaved PARP, Bcl-2, Bax, LC3B, AMPK, p-AMPKα(Thr172), mTOR, p-mTOR, p-4EBP, p70S6K, p-p70S6K, Atg5, Beclin1, ACC, p-ACC, CHOP, GRP78, GRP94, ATF6, PERK, p-PERK, IRE1α, p-IRE1α, eIF2α, p-eIF2α, cytosolic cyto c and GAPDH were purchased from Cell Signaling Technology (MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody was obtained from Santa Cruz Biotechnology.
Shen S., Zhang Y., Wang Z., Zhang R, & Gong X. (2014). Bufalin Induces the Interplay between Apoptosis and Autophagy in Glioma Cells through Endoplasmic Reticulum Stress. International Journal of Biological Sciences, 10(2), 212-224.
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6

Signaling Pathway Analysis by Western Blot

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Equal volumes of samples were loaded and proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane for Western blotting. Antibodies against LC3B, p62, phosphorylated ACCSer79 (pACC), phosphorylated AktSer473 (pAkt), phosphorylated AMPKThr172 (pAMPK), total AMPK, phosphorylated AS160Thr642 (pAS160), phosphorylated ERK1/2Thr202/Tyr204, phosphorylated HDAC5Ser498 (pHDAC5), phosphorylated mTORSer2448 (pmTOR), total mTOR, phosphorylated PKD1Ser744/748 (pPKD744), phosphorylated PKD1Ser916 (pPKD916), phosphorylated rS6Ser235/236 (prS6), phosphorylated TropISer23/24 (pTropI), and phosphorylated 4EBPThr37/46 (p4EBP) were used for detection. LC3B, p62, pAkt, pAMPK, pAS160, pmTOR, pPKD744, pPKD916, prS6, pTropI, p4EBP, and β-actin were obtained from Cell Signaling (Danvers, MA, USA). The antibody directed against pACC was from Upstate (Dundee, UK), pHDAC5 from Abcam (Cambridge, UK), and CAV3 from BD transduction Laboratories (San Jose, CA, USA). Membranes were blocked with 5% non-fat dry milk or 5% BSA in Tris-buffered saline with 0.1% Tween, incubated with primary antibodies overnight, and washed prior to incubation with HRP-conjugated secondary antibodies. Samples were normalized against the loading controls caveolin-3 (CAV3) or β-actin (β-actin). The protein bands were visualized using enhanced chemiluminescence (ClarityTM Western, Biorad, Hercules, CA, USA).
Geraets I.M., Coumans W.A., Strzelecka A., Schönleitner P., Antoons G., Schianchi F., Willemars M.M., Kapsokalyvas D., Glatz J.F., Luiken J.J, & Nabben M. (2021). Metabolic Interventions to Prevent Hypertrophy-Induced Alterations in Contractile Properties In Vitro. International Journal of Molecular Sciences, 22(7), 3620.
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7

Immunoblotting of Signaling Proteins

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Immunoblotting was performed as described [36] (link), using antibodies against rpS6 (#2217), phospho rpS6 (Ser235/236 [#2211] or Ser240/244 [#2215]), phospho S6K1(Thr389) [#9206], phospho Akt(ser473) [#4058], P-4E-BP(Thr37/46) [#2855], ß-actin [#4967], α-tubulin [#2144], mTOR [#2972], rictor [#2114] and raptor [#2280] (Cell Signaling Technology, Beverly, MA, USA), as well as FLAG (F3165, Sigma Sigma-Aldrich) and myc (SC-40, Santa Cruz). All antibodies were diluted 1∶1000. Exposures were chosen so that the chemiluminescent signals were within the linear response of the film and were quantified by ImageMaster VDS (Amersham Pharmacia Biotech).
Patursky-Polischuk I., Kasir J., Miloslavski R., Hayouka Z., Hausner-Hanochi M., Stolovich-Rain M., Tsukerman P., Biton M., Mudhasani R., Jones S.N, & Meyuhas O. (2014). Reassessment of the Role of TSC, mTORC1 and MicroRNAs in Amino Acids-Meditated Translational Control of TOP mRNAs. PLoS ONE, 9(10), e109410.
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8

Western Blot Analysis of Chondrocytes

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Cultured chondrocytes were washed twice in PBS and lysed in ice-cold RIPA buffer (Pierce) supplemented with protease and phosphatase inhibitor cocktail (Thermo Scientific). Twenty micrograms of protein were resolved in 4-12% acrylamide gels and transferred to nitrocellulose membranes using the Bolt system (Life Technologies). Membranes were washed in Tris-buffered saline (TBS) and blocked with Odyssey blocking buffer for 1 hour at room temperature. Then, blots were incubated overnight at 4°C with the following antibodies: REDD1 (1:500, Proteintech), p-S6 (1:2000, Cell Signaling), S6 (1:1000, Cell Signaling), p-4EBP (1:1000, Cell Signaling), 4EBP (1:1000, Cell Signaling), LC3 (1:1000, Cell Signaling), TXNIP (1:1000, Cell Signaling), and GAPDH (1:5000, Abcam). After washing three times with TBS with 0.05% Tween 20 (TBS-T), membranes were incubated with secondary antibodies goat anti-rabbit IRDye 800 (1:5000 dilution) and goat anti-mouse IRDye 680 (1:10000 dilution) for 1 hour at room temperature. Blots were washed three times in TBS-T and visualized using the Odyssey Infrared Imaging System (LI-COR). Intensity values were analyzed with the ImageStudioLite software and normalized to those of GAPDH.
Alvarez-Garcia O., Olmer M., Akagi R., Akasaki Y., Fisch K.M., Shen T., Su A.I, & Lotz M.K. (2016). Suppression of REDD1 in osteoarthritis cartilage, a novel mechanism for dysregulated mTOR signaling and defective autophagy. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society, 24(9), 1639-1647.
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9

Immunoblot Analysis of Autophagy-related Proteins

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The following antibodies were used: β‐Actin (Santa Cruz sc‐47778, 1:4,000), ULK1 (Cell signaling cat. 8054 1:1,000), Phospho‐ULK1 (Ser757) (Cell signaling cat. 6888 1:1,000), p70 S6 Kinase (Cell signaling cat. 2708 1:1,000), Phospho‐p70 S6 Kinase (Thr389) (Cell signaling cat. 9205 1:1,000), GAPDH (6C5) (Santa Cruz sc‐32233, 1:2,000), 4EBP (Cell signaling cat. 9644 1:1,000), p4EBP (Cell signaling cat. 9456 1:1,000), TFEB (Cell signaling cat. 4240S 1:1,000) and TFEB‐pS211 (custom‐generated in collaboration with Bethyl Laboratories 1:1,000). For immunoblot, the total cell lysates were prepared by solubilization of cell pellets in 10mM Tris–HCl pH 8.0 and 0.2% SDS supplemented with protease and phosphatase inhibitors (SIGMA). Protein concentration was determined by the Bradford method. After SDS‐PAGE and immunoblotting, the proteins recognized by the specific antibody were visualized by chemiluminescence methods (Luminata Crescendo Western HRP substrate, Millipore) using peroxidase‐conjugated anti‐rabbit or anti‐mouse secondary antibodies (Millipore). Membranes were developed using a Chemidoc UVP imaging system (Ultra‐Violet Products Ltd) and densitometric quantification was performed in unsaturated images using ImageJ (NIH).
Soldati C., Lopez‐Fabuel I., Wanderlingh L.G., Garcia‐Macia M., Monfregola J., Esposito A., Napolitano G., Guevara‐Ferrer M., Scotto Rosato A., Krogsaeter E.K., Paquet D., Grimm C.M., Montefusco S., Braulke T., Storch S., Mole S.E., De Matteis M.A., Ballabio A., Sampaio J.L., McKay T., Johannes L., Bolaños J.P, & Medina D.L. (2021). Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype. EMBO Molecular Medicine, 13(10), e13742.
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10

Western Blot Analysis of Murine Brain Proteins

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Mouse cortical tissue or isolated microglia were lysed by sonication in RIPA buffer supplemented with cOmplete and PhosSTOP. Samples were separated by 4–12% NuPAGE gels (Invitrogen, Cat# NP0335BOX) in MOPS buffer (Invitrogen, Cat# NP0001) and transferred to nitrocellulose membranes using the iBlot™ 2 Gel Transfer Device (Thermo Fisher Scientific). The membranes were blocked with 5% milk in 0.125% PBSX for 30min at RT, followed by incubation of primary antibodies overnight at 4°. The membranes were then washed with 0.0125% PBSX and incubated with HRP-conjugated secondary antibodies (Santa Cruz) for 1.5h at RT. Membranes were then washed and developed using the ChemiDoc™ MP Imaging System (BIO-RAD). Primary antibodies: HJ6.8B (for murine apoE, in house), α-tubulin (Sigma-Aldrich, Cat# T5168), LDLR (Abcam, Cat# ab52818), Lgmn (Santa Cruz, Cat# sc-133234), p4EBP (Cell signaling, Cat# 2855), actin (Cell signaling, Cat# 8456).
Shi Y., Andhey P.S., Ising C., Wang K., Snipes L.L., Boyer K., Lawson S., Yamada K., Qin W., Manis M., Serrano J.R., Benitez B.A., Schmidt R.E., Artyomov M., Ulrich J.D, & Holtzman D.M. (2021). Overexpressing low-density lipoprotein receptor reduces tau-associated neurodegeneration in relation to apoE-linked mechanisms. Neuron, 109(15), 2413-2426.e7.
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