P 4ebp1

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, China

P-4EBP1 is a primary antibody that specifically recognizes the phosphorylated form of eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1). 4EBP1 is an important regulator of protein synthesis and is phosphorylated by the mammalian target of rapamycin (mTOR) signaling pathway.

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256 protocols using p 4ebp1

Immunohistochemical Analysis of 4E-BP1 and p4E-BP1 in Prostate Cancer

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A total of 84 prostate cancer tissues and 22 precancerous tissues were obtained from the Ningbo Pathology Center of Zhejiang Province. Each case took 2–3 slides for experimentation. Slides were deparaffinized and hydrated at room temperature. Immunohistochemical staining was performed by following the manufacturer's directions of a specific kit (Absin, China). 4E‐BP1 (dilution 1:100; Cell Signaling, US) and p4E‐BP1 (dilution 1:100; Cell Signaling, US) were applied as the primary antibody at 37℃ for 12 h. For 4EBP1 or p4EBP1 scoring, the cytoplasmic staining intensity (4E‐BP1 or p4E‐BP1 typically stained the cytoplasmic of cancer cells) was rated as negative (no staining at all), weak (1+ staining intensity), moderate (2+ staining intensity), or strong (3+ staining intensity). In addition, it was stipulated that the product of the area percentage score of positive cells and the cell staining intensity score is regarded as a comprehensive staining score, with a score of ≥3 as positive, and a score of 0–2 as negative.

Wang K., Wang K, & Ma Q. (2022). The expression and significance of p4E‐BP1/4E‐BP1 in prostate cancer. Journal of Clinical Laboratory Analysis, 36(4), e24332.

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Compound C and Sodium Palmitate Signaling Pathway Analysis

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Compound C was obtained from Enzo Life Sciences. Sodium Palmitate is from NU-CHEK Prep Inc. For western blotting, anti-Flag M2 (RRID:AB_259529) antibody was purchased from Sigma, antibodies against SGK1 (RRID:AB_2687476), SGK2 (RRID:AB_10828732), SGK3 (RRID:AB_10949507), HSP90 (RRID:AB_2233307), Akt (RRID:AB_915783), p-Akt (Thr308) (RRID:AB_2255933), p-Akt (Ser473) (RRID:AB_2315049), NDRG1 (RRID: AB_11140640), p-NDRG1(Thr346) (RRID:AB_10693451), FoxO1 (RRID:AB_2106495), p-FoxO1/3 (Thr24/32) (RRID:AB_2106814), S6K (RRID:AB_390722), p-S6K (Thr389) (RRID:AB_2269803), S6 (RRID:AB_331355), p-S6 (S240/244) (RRID:AB_10694233), 4EBP1 (RRID:AB_2097841), p-4EBP1 (Thr37/46) (RRID:AB_560835), p-4EBP1 (Ser65) (RRID:AB_330947), AMPKα (RRID:AB_10624867), p-AMPKα (Thr172) (RRID:AB_331250), p-AMPKα(Ser485/491) (RRID:AB_331250), ACC1 (RRID:AB_2219397), p-ACC1 (Ser79) (RRID:AB_330337), Raptor (RRID:AB_561245)and p-Raptor (Ser792) (RRID:AB_2249475) were obtained from Cell Signaling Technology. Anti-actin (C4) was obtained from Abcam.

Zhou B., Zhang Y., Li S., Wu L., Fejes-Toth G., Naray-Fejes-Toth A, & Soukas A.A. (2021). Serum- and glucocorticoid-induced kinase drives hepatic insulin resistance by directly inhibiting AMP-activated protein kinase. Cell reports, 37(1), 109785.

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Immunoblotting Antibody Specifications

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Antibodies for immunoblotting such as p‐AMPKα (#2535), AMPKα (#5831), PDK1 (#3820), HIF1α (#3716), hexokinase 2 (#2867), LDHA (#3582), PFKP (#8164), PGAM1 (#12098), PDH (#3205), p‐Threonine/Tyrosine (#9381), p‐S6K (#9234), S6K (#2708), p‐ERK (#4370), ERK (#4695), p‐4E‐BP1 (Thr37/46, #2855), p‐4E‐BP1 (Ser65, #9451), 4E‐BP1 (#9644), mouse antirabbit IgG mAb (#5127), and antirabbit IgG (#7074) were from Cell Signaling Technology (Danvers, MA, USA). HIF1α (hydroxyl P564) (ab72777) and PHD3 (ab30782) were purchased from Abcam (Cambridge, MA, USA). HIF1α (hydroxyl P402) (07‐1585) was purchased from Merck (Darmstadt, Germany). GAPDH (sc‐25778) was purchased from Santa Cruz Biotechnology (Paso Robles, CA, USA). Actin (GTX109639) was purchased from GeneTex International (Hsinchu, Taiwan). Antibodies for immunoprecipitation such as HIF1α (#36169), rabbit mAb IgG (#3900), and p‐AMPKα (#2535) were purchased from Cell Signaling Technology. AMPKα1 (sc‐398861) was purchased from Santa Cruz Biotechnology. Antibodies for IHC such as p‐AMPKα (GTX52341) and HIF1α (GTX127309) were purchased from GeneTex International.

Tseng H., Zeng Y., Lin Y.J., Huang J., Lin C., Lee M., Yang F., Fang T., Mar A, & Su J. (2022). A novel AMPK activator shows therapeutic potential in hepatocellular carcinoma by suppressing HIF1α‐mediated aerobic glycolysis. Molecular Oncology, 16(11), 2274-2294.

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Comprehensive Antibody Validation for Western Blot and Immunofluorescence

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We used the following antibodies for western blotting: P‐AKT (S473; ref. 4060), P‐S6 (S240/244; ref. 5364), S6 (ref. 2217), AKT (ref. 9272), GSK‐3β (ref. 9315), P‐GSK‐3β (S9; ref. 9322), P‐4E‐BP1 (Ser65; ref. 9451), P‐4E‐BP1 (Thr37/46; ref. 2855), 4E‐BP1 (ref. 9644), RAPTOR (ref. 2280), RICTOR (ref. 2114), mTOR (ref. 2983), P‐AMPK (T172; ref. 2531), P‐ULK1 (Ser757; ref. 6888), SDH (ref. 11998) from Cell Signalling, LC3 (ref. L7543) and p62 (ref. P0067) from Sigma, GAPDH (ref. 8245), Mitoprofile (ref. 110413) and TOM20 (ref. 56783) from Abcam, actin (ref. 56459), and GRP‐75 (ref. 13967) and Porin1 (ref. 390996) from Santa Cruz. For immunofluorescence, LAMP1 (ref. 1D4B), embryonic MyHC (ref. BF‐G6), and types I, IIa, and IIb MyHC (ref. BAD5, SC‐71, and BF‐F3, respectively) were from Developmental Studies Hybridoma Bank, mTOR was from Cell Signalling, distrophin (ref. 15277) from Abcam, NCAM (ref. 5032) was from Millipore, and α‐bungarotoxin Alexa Fluor 555‐conjugate (ref. B35451) was from Invitrogen.

Baraldo M., Geremia A., Pirazzini M., Nogara L., Solagna F., Türk C., Nolte H., Romanello V., Megighian A., Boncompagni S., Kruger M., Sandri M, & Blaauw B. (2019). Skeletal muscle mTORC1 regulates neuromuscular junction stability. Journal of Cachexia, Sarcopenia and Muscle, 11(1), 208-225.

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Intestinal Mucosal Protein Quantification

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The Western blot was used to detect liver kinase B1 (LKB1), ras homolog enriched in the brain (Rheb), tuberous sclerosis complex 2 (TSC2), Raptor, mTOR, S6K1, and 4EBP1 phosphorylation and expression levels of total proteins in jejunal and ileal mucosa, respectively. The jejunal and ileal mucosa samples were homogenized, and protein concentrations were measured using the bicinchoninic acid assay method with BSA as standard (Catalog# P0010, Beyotime Institute of Biotechnology, Shanghai, China). The following first antibodies were used for protein quantification: LKB1 (1:1,000, Cell Signaling Technology); Raptor (1:1,000, Cell Signaling Technology); Rheb (1:1,000, Cell Signaling Technology); mTOR (1:1,000, Cell Signaling Technology); TSC2 (1:1,000, Cell Signaling Technology); S6K1 (1:1,000, Cell Signaling Technology); 4EBP1 (1:1,000, Cell Signaling Technology); p-mTOR (1:1,000, Cell Signaling Technology); p-TSC2 (1:1,000, Cell Signaling Technology); p-S6K1 (1:1,000, Cell Signaling Technology); and p-4EBP1 (1:1,000, Cell Signaling Technology) and p-4EBP1(1:1,000, Cell Signaling Technology).

Deng Y., Cheng H., Li J., Han H., Qi M., Wang N., Tan B., Li J, & Wang J. (2023). Effects of glutamine, glutamate, and aspartate on intestinal barrier integrity and amino acid pool of the small intestine in piglets with normal or low energy diet. Frontiers in Veterinary Science, 10, 1202369.

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Compound C and Sodium Palmitate Signaling Pathway Analysis

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Immunological Reagents and Assays for mTOR Pathway

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Immunological reagents were obtained from the following sources (Cat # in parenthesis): Tor1-specific antibody was previously described (Li et al., 2006 (link)). HRP-labeled secondary antibodies, Santa-Cruz; Antibodies for mTOR (2983), Raptor (2280), Rheb (4935), RagA (4537), RagB (8150), RagC (5466), RagD (4470), Lamp1 (9091), Rictor, P-S6K1(T398), S6K1, P-AKT(S473), AKT, P-4E-BP1(T37/46), 4E-BP1, ERK, P-ERK(T202/Y204), α-tubulin and Myc-epitope, Cell Signaling Technology; Antibody for HA-epitope, Bethyl Laboratories; Antibodies for Rab1A and P-S6K1(T398) for IHC, Lamp2 antibody (ab25630), Abcam; Antibody for Rab1A (11671 for WB and IP), Rab1B, Proteintech Group; Rab1A antibody for IF (H00005861-M07A), Abnova; Protein G-Sepharose, GE Healthcare; EDTA-free Complete Protease Inhibitor Cocktail and PhosSTOP, Roche; rapamycin and PD98059, Selleck Chemicals. Cell lysis and Western blot (Drenan et al., 2004b (link); Sancak et al., 2008 (link)) and immunoprecipitation (Sancak, et al, 2008 (link)) were performed as described previously. For determining if Ratpor or mTOR mediates the interaction with Rab1A, anti-Rab1A immunoprecitates were washed with cell lysis buffer containing 0.25% or 0.50% TX-100.

Thomas J.D., Zhang Y., Wei Y., Cho J.H., Morris L.E., Wang H.Y, & Steven Zheng X. (2014). Rab1A Is an mTORC1 Activator and a Colorectal Oncogene. Cancer cell, 26(5), 754-769.

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Evaluating SNX-2112 Cytotoxicity and Signaling

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SNX-2112 was synthesized as previously described in our lab with >98.0% purity [42 (link)], dissolved in Dimethyl sulfoxide (DMSO) to obtain a 100 mM stock solution, and stored at −20°C. TRAIL was purchased from Merck Millipore (Waltham, MA, USA). N-Acetyl-cysteine (NAC) and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Bafilomycin A1 (BFA) was purchased from Selleck Chemicals (Shanghai, China). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). Antibodies for GAPDH, Bcl-2, Bcl-xL, FLIP, pro-caspase 3, cleaved-caspase 3 (c-caspase 3), cleaved-caspase 8 (c-caspase 8), cleaved-PARP (c-PARP), Akt, p-Akt (Ser473), DR4, DR5, LC3, Beclin1, Atg7, p62, p-mTOR, p-S6, p-4EBP1, p53, p-ERK, ERK, p-p38, p38, p-JNK, and JNK were purchased from Cell Signaling Technology (CST; Beverly, MA, USA).

Hu L., Wang Y., Chen Z., Fu L., Wang S., Zhang X., Zhang P., Lu X., Jie H., Li M., Wang Y, & Liu Z. (2019). Hsp90 Inhibitor SNX-2112 Enhances TRAIL-Induced Apoptosis of Human Cervical Cancer Cells via the ROS-Mediated JNK-p53-Autophagy-DR5 Pathway. Oxidative Medicine and Cellular Longevity, 2019, 9675450.

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Western Blot Analysis of Cell Signaling

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Western blot protein assays were conducted as previously reported [20 ]. Cells were treated with omipalisib and harvested by trypsinization, followed by resuspension in lysis buffer (containing 1 mM PMSF and 0.5 mM PhosSTOP) with brief sonication. The protein concentrations were determined with a protein assay kit according to the manufacturer’s instructions (Beyotime, P0010, China). Cell lysates were separated by SDS-PAGE and transferred onto PVDF membranes (GE Healthcare). After blocking with blocking buffer containing 5% nonfat milk, membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase–conjugated secondary antibody. Blots were imaged with an imaging system (MYECL, Thermo scientific). Primary antibodies, AKT (#9272), ERK (#4695), P70S6K (#9202), S6 (#2217), 4EBP1 (#9644), p-AKT (Ser473, #4060), p-ERK (Thr202/Tyr204, #4376), p-P70S6K (Ser371, #9208), p-S6 (Ser235/236, #2211), p-4EBP1 (Thr37/46, #2855), and CDKN1B (#3688) were purchased from Cell Signaling Technology. CCND1 (ab134175) was obtained from Abcam.

Zhu D.S., Dong J.Y., Xu Y.Y., Zhang X.T., Fu S.B, & Liu W. (2020). Omipalisib Inhibits Esophageal Squamous Cell Carcinoma Growth Through Inactivation of Phosphoinositide 3-Kinase (PI3K)/AKT/Mammalian Target of Rapamycin (mTOR) and ERK Signaling. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e927106-1-e927106-13.

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TNBC Cell Lines Characterization

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Human TNBC cell lines: BT-549, HCC-1806, HCC-1937, MDA-MB-231, MDA-MB-453, MDA-MB-468 and SUM-159 were obtained from the American Type Culture Collection (ATCC) and were maintained using ATCC recommended media. All model cells utilized were free of mycoplasma contamination. Additionally, STR DNA profiling was used to confirm the identity of cell lines. The GAPDH, p-ERK (42/44), ERK (42/44), p-S6 (s235/236), S6, p-mTOR (S2448), mTOR, p-4EBP1, 4EBP1 and c-Myc antibodies were obtained from Cell Signaling Technology (Beverly, MA). LAS1L, TEX-10, SENP3 antibodies were purchased from Proteintech (Rosemont, IL). The β-Actin antibody (A-2066), WDR18 antibody (HPA050200) and Vinculin antibody (V9264) were purchased from Millipore Sigma (Burlington, MA). The Ki67 antibody (ab1667) was purchased from Abcam (Cambridge, MA). The PELP1 antibody (A300-180A) was purchased form Bethyl Laboratories Inc. (Montgomery, TX).

Altwegg K.A., Pratap U.P., Liu Z., Liu J., Sanchez J.R., Yang X., Ebrahimi B., Panneerdoss D.M., Li X., Sareddy G.R., Viswanadhapalli S., Rao M.K, & Vadlamudi R.K. (2023). Targeting PELP1 oncogenic signaling in TNBC with the small molecule inhibitor SMIP34. Breast Cancer Research and Treatment, 200(1), 151-162.

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