P 4ebp1 s65

Manufactured by Cell Signaling Technology

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P-4EBP1 (S65) is an antibody that detects the phosphorylation of 4EBP1 at serine 65. 4EBP1 is a protein that regulates the activity of the eukaryotic translation initiation factor 4E (eIF4E), which is involved in the initiation of protein synthesis. Phosphorylation of 4EBP1 at serine 65 is an important regulatory event that modulates its interaction with eIF4E.

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P-4EBP1 (256 citations)

19 protocols using p 4ebp1 s65

Immunoblotting of Cell Signaling Proteins

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The following primary antibodies from Cell Signalling Technology (Danvers, MA, USA) were used: 4E‐BP1 (#9452), P‐4E‐BP1^S65 (#9451), AKT (#9272), P‐AKT^S473 (#9271), P‐AKT^T308 (#9275), eIF4E (#2067), mTOR (#2972), P‐mTOR^S2448 (#2971), raptor (#2280), S6 (#2217), and S6^S240/244 (#5364). The primary antibody for α‐actinin (#A7732) was purchased from Abcam (Cambridge, England). The primary antibody for EGFP (11814460001) was from Roche Applied Science (Penzberg, Germany). All Cell Signalling Technology antibodies as well as EGFP were diluted 1:1000; α‐actinin was diluted 1:5000. The secondary antibodies goat anti‐mouse (#115‐035‐003) and goat anti‐rabbit (#111‐035‐003) were purchased from Jackson ImmunoResearch Europe (Ely, Cambridgeshire, England) and were diluted 1:10 000. All antibodies were diluted in 4% bovine serum albumin in TBS‐T.

Ham A.S., Chojnowska K., Tintignac L.A., Lin S., Schmidt A., Ham D.J., Sinnreich M, & Rüegg M.A. (2019). mTORC1 signalling is not essential for the maintenance of muscle mass and function in adult sedentary mice. Journal of Cachexia, Sarcopenia and Muscle, 11(1), 259-273.

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Immunoblotting Analysis of Protein Phosphorylation

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Cells were washed with PBS once, disrupted on ice for thirty minutes in NP-40 (50 mM Tris [pH 7.4], 1% NP-40, 150 mmol/L NaCl, 40 mmol/L NaF) or RIPA lysis buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitors (Pierce Chemical) and cleared by centrifugation. Protein concentration was determined with BCA reagent from Pierce. Equal amounts of protein (10 to 50 µg) in cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes (GE healthcare), immunoblotted with specific primary and secondary antibodies and detected by chemiluminescence with the ECL detection reagents from Amersham Biosciences. Antibodies for P-AKT (S473) (#4060L), P-p70S6K (T389) (#9234L), P-S6 (S240/244) (#5364L) and P-S6 (S235/236) (#4858L), P-4EBP1 (T37/46) (#9459L), P-4EBP1 (S65) (#9451L), P-4EBP1 (T70) (#9455L), β-actin (#4970S), mTOR (#2972S) and Raptor (#2280S) were purchased from Cell Signaling Technology. The FLAG (#F1804) antibody was purchased from Sigma. The GST antibody (#sc-138) was from Santa Cruz.

Rodrik-Outmezguine V.S., Okaniwa M., Yao Z., Novotny C.J., McWhirter C., Banaji A., Won H., Wong W., Berger M., de Stanchina E., Barratt D.G., Cosulich S., Klinowska T., Rosen N, & Shokat K.M. (2016). Overcoming mTOR Resistance Mutations with a New Generation mTOR Inhibitor. Nature, 534(7606), 272-276.

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Immunoblotting Analysis of Signaling Pathways

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Cells were treated with or without C96 for 24 hrs before being applied to lysate preparation in a lysis buffer (50 mM Tris-HCI, pH 7.4, 1% NP-40, 0.5% Na-deoxycholate, and 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 2 mM Na₃VO₄, and 5 mM NaF) [6 (link)]. Equal amounts (30 μg) of total proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) separation, followed by immunoblotting analyses with specific antibodies. Antibodies against PARP, caspase-3, AKT, p-AKT(S473), 4E-BP1, p-4E-BP1(S65), IGF-1R, p-IGF-1R, ERK(p42/p44), and p-ERK(p42/p44) were purchased from Cell Signaling Technologies. Antibodies against mTOR, p-mTOR(S2448), p70S6K, p-p70S6K(S424), and GAPDH were obtained from Abgent (Suzhou, China). Horseradish peroxidase-conjugate secondary Antibodies against mouse or rabbit and GAPDH were purchased from Abgent. All immunoblotting signals were further analyzed with Quality One Quality One software (Bio-Rad, Berkeley, CA, USA).

Tang J., Zhu J., Yu Y., Zhang Z., Chen G., Zhou X., Qiao C., Hou T, & Mao X. (2014). A virtual screen identified C96 as a novel inhibitor of phosphatidylinositol 3-kinase that displays potent preclinical activity against multiple myeloma in vitro and in vivo. Oncotarget, 5(11), 3836-3848.

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Comprehensive Antibody Reagents for Cellular Signaling Analysis

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Antibodies against pS6K (T389) (#9205), S6K (#9202), pS6 (S240/244) (#2215), S6 (#2217), pAkt (T308) (#13038), pAkt (S473) (#4060), AKT (4691), p 4E-BP1 (S65) (#9456), 4E-BP1 (#9644), RagA (#4357), RagB (#8150), RagC (#9480), Sestrin2 (#8487), MIOS (#13557), EEA1 (#3288, used for immunofluorescence (IF)), Lamp1 (#9091, used for IF) and Rab7 (#9367, used for IF) were purchased from Cell Signaling Technology. Lamp2 mouse monoclonal antibody (H4B4, for human cells) and Lamp2 rat-monoclonal antibody (GL2A7, for mouse cells) for IF were obtained from Abcam. Anti-FLAG (F1804, used for IF), anti-HA (3F10), and anti-PMP70 (SAB4200181, used for IF) were obtained from Sigma. WDR24 (20778-1-AP, used for IP) and Sestrin2 (10795-1-AP, used for IP) antibody was purchased from Proteintech. Antibodies against SZT2 (sc-242153), NPRL2 (sc-376986), SEC13 (sc-514308) and WDR59 (sc-137927, only used in immunoblotting of WDR59 in MEFs) were obtained from Santa Cruz. SEH1L (ab187307) antibody was form Abcam. The rabbit WDR59 monoclonal antibody used in IF experiments (1:100, validated in this study with knockout cells, Extended Data Fig. 5b) and immunoblotting experiments (1:1000) in HEK293T was kindly provided by Jianxin Xie from Cell Signaling Technology. This antibody only recognized WDR59 from human cells, but not MEFs. Anti-NPRL3 (NBP1-88447, used for IP) was obtained from Novus Biologicals.

Peng M., Yin N, & Li M.O. (2017). SZT2 Dictates GATOR Control of mTORC1 Signaling. Nature, 543(7645), 433-437.

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Western Blot Analysis of Insulin Signaling

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Typically, 10 µg of protein was resolved by SDS–PAGE, transferred to PVDF membranes and immunoblotted as previously described [5 (link)]. Antibodies detecting TRARG1 (sc-292062 or sc-377025) and 14-3-3 (sc-1657) were from Santa Cruz Biotechnology. Antibodies against pHSL S660 (#4126), pp90RSK (#9344), p4EBP1 S65 (#9456), pTBC1D4 T642 (#4288S), pAKT S473 (#4051), pAKT T308 (#9275L), AKT (#4685), HA (#C29F4), GSK3α (#9338S), GSK3β (#9832S), pGSK3 Ser 9/21 (#8566S), pGS S641 (#3891) and Caveolin1 (#3267) were purchased from Cell Signaling Technology. Anti-α-tubulin (#T9026) was from Sigma–Aldrich. Antibody against TBC1D4 was produced as previously described [4 (link)].

Duan X., Norris D.M., Humphrey S.J., Yang P., Cooke K.C., Bultitude W.P., Parker B.L., Conway O.J., Burchfield J.G., Krycer J.R., Brodsky F.M., James D.E, & Fazakerley D.J. (2022). Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate. Biochemical Journal, 479(11), 1237-1256.

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Muscle Protein Analysis by Western Blot

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Muscle sample preparation and western blot analysis were performed as described previously (26 (link)). The following primary antibodies were used: LC3 (Novus Biological NB100–2220), p62/SQSTM1 (sigma P0067), Atg6 (Novus Biological NB500–249), Atg7 (R&D systems MAB6608), Puromycin (Kerafast EQ001), Akt (Cell signaling CST9272), p-Akt (S473) (Cell signaling CST9271), Raptor (Cell signaling CST2280), p-p70S6k (T389) (Cell signaling CST9205), p70S6k (Abcam AB37699), p-4e-bp-1 (S65) (Cell signaling CST9451), 4e-bp-1 (Cell signaling CST9452), Cox4 (Abcam AB33985), OXPHOS cocktail (Thermo PA5–28220), and Gapdh (Cell signaling CST2118).

Cui D., Drake J.C., Wilson R.J., Shute R.J., Lewellen B., Zhang M., Zhao H., Sabik O.L., Onengut S., Berr S.S., Rich S.S., Farber C.R, & Yan Z. (2020). A novel voluntary weightlifting model in mice promotes muscle adaptation and insulin sensitivity with simultaneous enhancement of autophagy and mTOR pathway. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(6), 7330-7344.

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Cellular Senescence Assay Protocol

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AOA, H₂O₂, NAC, αKG, dimethylα-ketoglutarate (dmαKG), sodium pyruvate, oxaloacetate and 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) were purchased from Sigma-Aldrich. Rapamycin and Torin1 were purchased from Tocris Bioscience (Minneapolis, USA). Stock solutions of AOA (0.5 M), αKG (0.5 M), pyruvate (0.5 M), oxaloacetate (0.5 M) and NAC (0.5 M) were prepared in ddH₂O, sterilized by filtration and then stored as aliquots at −20°C. Rapamycin and Torin1 were dissolved initially in DMSO to 1 mM and 750 µM stock solutions, respectively. DCFDA was freshly prepared in DMSO at 5 mM. The antibodies against S6K1 (#9202), P-S6K-T389 (#9205), 4E-BP1 (#9452), P-4E-BP1-S65 (#9451), AKT (#9272) and P-AKT-S473 (#9271) were purchased from Cell Signaling Technology; the antibodies against pRb (#IF8), p53 (#DO1), p21^CIP1 (#F5) and p16^INK4A (#H156) were from Santa Cruz Biotechnology; another anti-p16 (ab81278) and anti-H3K9Me3 (ab8898) antibodies were from Abcam (Cambridge, UK); anti-β-actin was from Sigma-Aldrich; and all HRP-conjugated secondary antibodies were from Pierce Thermo Fisher Scientific.

Liao G.Y., Lee M.T., Fan J.J., Hsiao P.W., Lee C.S., Su S.Y., Hwang J.J, & Ke F.C. (2019). Blockage of glutamine-dependent anaplerosis affects mTORC1/2 activity and ultimately leads to cellular senescence-like response. Biology Open, 8(5), bio038257.

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Immunoblotting Assay for Signaling Proteins

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The immunoblotting was conducted as before [12] (link). The primary antibodies used were presented as follows, FLT3, p-AKT (Ser473), AKT, p-ERK (T202/Y204), Caspase-3, PARP, 4E-BP1 and p4E-BP1 (S65), which were all purchased from Cell Signaling Technology (Beverly, MA). ERK1/2, STAT5, p-STAT5 (Y694) and β-actin were obtained from Huabio (Hangzhou, China). p-FLT3 (Y591), MCL-1, BCL-XL, BCL-2, CDK-2, CDK-4 and CDK-6 were purchased from Abcam (Cambridge, MA).

Wu Z., Zhuang H., Yu Q., Zhang X., Jiang X., Lu X., Xu Y., Yang L., Wu B., Ma A., Zhang L., Xiao X., Liang Y., Gao R., Shen J, & Xu R. (2019). Homoharringtonine Combined with the Heat Shock Protein 90 Inhibitor IPI504 in the Treatment of FLT3-ITD Acute Myeloid Leukemia. Translational Oncology, 12(6), 801-809.

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Profiling mTOR Pathway Activation

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Antibodies used include UCH-L1 (3524), raptor (2280), rictor (9476), 4EBP1 (2845), p4EBP1^T70 (5078), p4EBP1^T37/46 (2855), p4EBP1^S65 (9451), pS6K (9208), S6K (49D7), AKT (4691), pAKT^S473 (4060), eIF4A (C32B4), eIF4E (C46H6), anti-DYKDDDDK Tag (8146), anti-MYC tag (9B11) from Cell Signaling Inc. (Danvers, MA, USA), anti-HA (3F10, Roche Applied Science, Indianapolis, IN, USA) tubulin (T9026) from Sigma, mTOR (A301-143a), PHLPP (A300-660A) from Bethyl Laboratories (Montgomery, TX, USA), eIF4G (ab31217) from Abcam. Immunoprecipitations were performed as described using lysates prepared with 0.3% CHAPS (mTOR/rictor/raptor)^{4 (link),30 (link)}, 0.5% NP40 (BioID2-UCHL1, UCHL1-HA) or 1% SDS (raptor for detection of FLAG-Ub)^{4 (link)}. Immobilized m⁷GTP (AC-155) was from Jena Bioscience (Jena, Germany). M⁷GTP pulldowns were performed as described^{3 (link)}.

Hussain S., Bedekovics T., Ali A., Zaid O., May D.G., Roux K.J, & Galardy P.J. (2019). A cysteine near the C-terminus of UCH-L1 is dispensable for catalytic activity but is required to promote AKT phosphorylation, eIF4F assembly, and malignant B-cell survival. Cell Death Discovery, 5, 152.

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Western Blot Analysis of Cellular Proteins

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Cells were washed and scraped in PBS, centrifuged and the pellet was lysed using RIPA buffer containing protease and phosphatase inhibitor cocktail (Thermo Scientific, Cat. # 78440). 25–30μg of protein was used for SDS PAGE. Tumor tissues were dissociated in RIPA buffer with protease and phosphatase inhibitor cocktail using a pellet pestle followed by sonication for 10 minutes. All primary antibodies were diluted in 5% milk in Tris-buffered Saline/0.1% Tween20 and incubated overnight at 4°C. The following primary antibodies were used: PHGDH (Sigma, Cat. #HPA021241), ATF4 (Cell Signaling Technologies, Cat. #11815S), pERK (T202/204) (Cell Signaling Technologies, Cat. # 9101S), ERK (Cell Signaling Technologies, Cat. # 4695S), p S6 Ribosomal protein (S235/236) (Cell Signaling Technologies, Cat. # 4858S), S6 Ribosomal protein (Cell Signaling Technologies, Cat. # 2217S), p 4EBP1 (S65) (Cell Signaling Technologies, Cat. # 9451S), 4EBP1 (Cell Signaling Technologies, Cat. # 9452S), HA-Tag (Cell Signaling Technologies, Cat. # 3724S), pEIF2α (S51) (Cell Signaling Technologies, Cat. # 3398), EIF2a (Cell Signaling Technologies, Cat. # 5324), GFP (Cell Signaling Technologies, Cat. # 2956S), BRAF (Sigma, Cat. # HPA001328) and β-actin (Invitrogen, Cat. # AM4302)

Jasani N., Xu X., Posorske B., Kim Y., Vera O., Tsai K.Y., DeNicola G.M, & Karreth F.A. (2024). MAPK-mediated PHGDH induction is essential for melanoma formation and represents an actionable vulnerability. bioRxiv.

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