Eif4g

Manufactured by Cell Signaling Technology

Sourced in United States

EIF4G is a protein involved in the initiation of protein translation. It functions as part of the eukaryotic translation initiation complex, playing a crucial role in the recruitment of the ribosome to the mRNA.

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Anti-eIF4G (11 citations) Rabbit anti-eIF4G (6 citations) P-eIF4G (5 citations) EIF4G (2858) (2 citations) EIF4G (Ser1108) (2 citations) Rabbit anti-eIF4G antibody (2 citations)

35 protocols using eif4g

Western Blot Analysis of Protein Expression

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Protein samples were prepared in reduced and denatured forms (32 (link)) and resolved using SDS-PAGE. The proteins were transferred to a nitrocellulose membrane and then blocked with 5% nonfat milk in TBS-T (0.02 M Tris–HCl, 0.16 M NaCl, and 0.1% Tween-20, pH 7.4), at room temperature, for 1 h. The membranes were incubated overnight with primary antibodies PABPC4 (Bethyl, #A301-466A), NCoR1 (Affinity, #AF0270), PABPC1 (Thermo Fisher Scientific, #PA5-29883), FLAG (Sigma-Aldrich, #F1804), α-tubulin (Sigma-Aldrich, #T9026), OXPHOS (proteins of mitochondrial ETC) (Abcam, #ab110413), PPARD (Thermo Fisher Scientific, #PA1-823A), eIF4G (Cell Signaling Technology, #2498), puromycin (Merck, #MABE343), Vinculin (Cell Signaling Technology, #4650), Lamin A (Santa Cruz Biotechnology, #sc-71481), and β actin (Santa Cruz Biotechnology, #81178), Akt (Cell Signaling Technology, #9272), p-Akt^Thr308 (Cell Signaling Technology, #9275), ubiquitin (Abcam, #ab7254), and GST (Sigma-Aldrich, #G7781). The membrane was then washed with TBS-T and incubated with horseradish peroxidase–conjugated secondary antibody (1:10,000) in TBS-T solution containing 5% nonfat for 1 h. Membranes were washed with TBS-T and then added the peroxidase substrate SuperSignal West Plus (Thermo Fisher Scientific), and the band intensities were captured in the ImageQuant LAS500 (GE Healthcare).

Oliveira A.G., Oliveira L.D., Cruz M.V., Guimarães D.S., Lima T.I., Santos-Fávero B.C., Luchessi A.D., Pauletti B.A., Leme A.P., Bajgelman M.C., Afonso J., Regitano L.C., Carvalho H.F., Carneiro E.M., Kobarg J., Perissi V., Auwerx J, & Silveira L.R. (2023). Interaction between poly(A)–binding protein PABPC4 and nuclear receptor corepressor NCoR1 modulates a metabolic stress response. The Journal of Biological Chemistry, 299(6), 104702.

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Comprehensive Western Blot Analysis

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Cells were lysed in RIPA buffer (Beyotime, Jiangsu, China) containing a protease inhibitor cocktail (Sigma, St. Louis, CA, USA). Total protein was assessed using a BCA Protein Assay Kit (Beyotime). Western blot analysis was performed as previously described [18 (link)]. Antibodies against the following were used in this study, PABPC1 (1:1000, Abcam, SF, USA), IFI27, HA tag, Flag tag, TSG101, CD63, CD9, PCNA, Alix, Ki67, caspase 3, CXCL10, CD34, cleaved-PARP, PARP, eIF4G, cleaved caspase 9, caspase 9, ERK, p-ERK, IFI27, STAT3, p-STAT3, STAT1, p-STAT1, NF-kB, p-NF-kB, β-actin and GAPDH (all 1:1000, Cell Signaling Technology, MA, USA), EXOSC2 and EXOSC4 (both 1:1000, Santa Cruz, MA, USA).

Zhang Y., Chen C., Liu Z., Guo H., Lu W., Hu W, & Lin Z. (2022). PABPC1-induced stabilization of IFI27 mRNA promotes angiogenesis and malignant progression in esophageal squamous cell carcinoma through exosomal miRNA-21-5p. Journal of Experimental & Clinical Cancer Research : CR, 41, 111.

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Antibody Profiling of mTOR Pathway

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The following antibodies were used in this study: 4EBP1 (catalog #9644, 1:4000 dilution), Phospho-4EBP1-Thr37/46 (#2855,1:1000), Phospho-4EBP1 -Ser65 (#9451,1:1000), p70S6 Kinase (#2708,1:1000), Phospho-p70S6 Kinase-Thr389 (#9234,1:1000), Phospho-S6-Ser240/244(#5364,1:2000), AKT(#4691,1:2000), Phospho-AKT-Ser473 (#4060,1:1000), EIF4E (#9742,1: 2000), EIF4G (#2498,1:1000), NDRG1(#5196,1:2000), Phospho-NDRG1-Thr 346(#5482,1:2000), Phospho-Tuberin/TSC2-Ser939 (#3615,1:1000), SGK3 (#85731:1000), Phospho-SGK3-Thr320 (#5642,1:500), Survivin (#2808, 1:1000) and Rictor (#2114,1:1000), and were purchased from Cell Signaling Technology (Danvers, MA). In addition, γ-Tubulin (sc-7396,1:500) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Tuberin (ab32554,1:1000) and INPP4B (ab81269, 1:1000) were purchased from Abcam (Cambridge, UK).

Wang H., Huang F., Zhang Z., Wang P., Luo Y., Li H., Li N., Wang J., Zhou J., Wang Y, & Li S. (2019). Feedback Activation of SGK3 and AKT Contributes to Rapamycin Resistance by Reactivating mTORC1/4EBP1 Axis via TSC2 in Breast Cancer. International Journal of Biological Sciences, 15(5), 929-941.

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Western Blot Analysis of Key Signaling Proteins

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The total cells were treated in lysis buffer [150 mM NaCl, 10 mM Tris (pH 7.2), 5 mM EDTA, 0.1% Triton X-100, 5% glycerol, and 2% SDS]. Each amount of the protein extracts were denatured by boiling at 95 °C for 10 min in sample buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The total proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking at room temperature for 1 h using PBST containing 10% BSA with gentle shaking, the membranes were incubated with primary antibodies (1:1000) overnight at 4 °C with gentle shaking, incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.), and then visualized with the enhanced chemiluminescence (ECL) detection system (GE Healthcare, Piscataway, NJ, USA). All of the primary antibodies (mTOR, PDK1, PRAS40, 4EBP-1, P70S6K, S6, Raptor, Rictor, eIF4E, eIF4G, HIF-1α, Cyclin D1, C-myc, Bcl2, VEGF, PARP, Caspase-3, Tubulin, GAPDH and β-actin) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Liu J., Liu Y., Zhang J., Liu D., Bao Y., Chen T., Tang T., Lin J., Luo Y., Jin Y, & Zhang J. (2020). Indole hydrazide compound ZJQ-24 inhibits angiogenesis and induces apoptosis cell death through abrogation of AKT/mTOR pathway in hepatocellular carcinoma. Cell Death & Disease, 11(10), 926.

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Western Blot Analysis of Translation Regulators

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Western blot was described previously [6 (link)]. Briefly, cells were lysed using Laemmli buffer containing 10% dithiothreitol (DTT, Thermo Fisher Scientific, Carlsbad, USA), and then denatured by heating at 95 °C for 5 min. Proteins were detected with antibodies against eIF4A (Cell Signaling Technology, Danvers, USA), eIF4E (Cell Signaling Technology), eIF4G (Cell Signaling Technology), PDCD4 (Cell Signaling Technology), and SA11 rotavirus VP4 (provided by professor Harry Greenberg, Stanford University School of Medicine, USA). The bound antibodies were visualized with Odyssey (LI-COR Biosciences, USA). The β-actin (Santa Cruz) was used as a loading control.

Chen S., Feng C., Fang Y., Zhou X., Xu L., Wang W., Kong X., P. Peppelenbosch M., Pan Q, & Yin Y. (2019). The Eukaryotic Translation Initiation Factor 4F Complex Restricts Rotavirus Infection via Regulating the Expression of IRF1 and IRF7. International Journal of Molecular Sciences, 20(7), 1580.

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Quantifying Muscle Protein Signaling

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The gastrocnemius muscle samples were homogenised in buffer containing 20 mM Tris, 1 mM DTT, 2 mM ATP, and 5 mM MgCl₂, centrifuged at 12,000 g, and total protein content was assessed using the Lowry method [32 (link)]. Muscle samples (60 µg of protein) were analysed using SDS-polyacrylamide gel electrophoresis (10% or 12%) and transferred to a 0.45-µm pore size nitrocellulose membrane, which was blocked with skim milk (5%) for 1 h. Proteins were probed with primary antibodies GAPDH (SC47724) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); 20S (PW8195), 19S (PW9265), and 11S (PW8185) (Enzo Life Sciences, Farmingdale, NY, USA); PI3K (4292), phosphor-PI3K (4228), mTOR (2972), phospho-mTOR (2971), p70S6K (9202), phospho-p70 S6KThr421/Ser424 (9204), 4E-BP1 (9452), phospho-4E-BP1Thr70 (9455), and eIF4G (2498) (Cell Signalling, Danvers, MA USA); and secondary antibodies goat anti-rabbit (7074) and horse anti-mouse (7076) (Cell Signalling). The Western blot band images were captured using the Alliance 2.7 (UVITEC, Cambridge, UK) and quantified using UVIband-1D (UVITEC), and protein expression was normalised using GAPDH as a loading control.

Miyaguti N.A., de Oliveira S.C, & Gomes-Marcondes M.C. (2019). Maternal Leucine-Rich Diet Minimises Muscle Mass Loss in Tumour-bearing Adult Rat Offspring by Improving the Balance of Muscle Protein Synthesis and Degradation. Biomolecules, 9(6), 229.

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Protein Expression Profile Analysis

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Anti-puromycin (Kerafast EQ0001), pAKT S473 (Cell Signaling Technology (CST) 4060), p 4EBP1 T37/46 (CST 2855), Cyclin D1 (CST 55506), Estrogen Receptor Alpha N-terminus (CST 13258), Estrogen Receptor Alpha C-terminus (CST 8644), Progesterone Receptor (CST 8757), EIF4G (CST 2498), EIF4E (CST 9742), 4EBP1 (CST 9644), Beta Actin (CST 4967), Myc (CST 18583), GATA3 (CST 5852), Cyclin D3 (CST 2936), CDK4 (CST 12790), pRb S780 (CST 9307), E2F1 (CST 3742), GREB1 (CST 65171), TFF1/pS2 (CST 15571), IGFBP4 (CST 31025), cleaved PARP (CST 5625). Secondary Goat anti-Rabbit IgG (H+L) Secondary Antibody HRP (Thermo Fisher 65–6120).

Boyer J.A., Sharma M., Dorso M.A., Mai N., Amor C., Reiter J.M., Kannan R., Gadal S., Xu J., Miele M., Li Z., Chen X., Chang Q., Pareja F., Worland S., Warner D., Sperry S., Chiang G.G., Thompson P.A., Yang G., Ouerfelli O., de Stanchina E., Wendel H.G., Rosen E.Y., Chandarlapaty S, & Rosen N. (2024). eIF4A controls translation of estrogen receptor alpha and is a therapeutic target in advanced breast cancer. bioRxiv.

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Western Blot Analysis of Key Signaling Proteins

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Western analysis was performed as previously described [18] (link). The following antibodies were used for immunoblotting: eIF4E, 4E-BP1, eIF4G, 4EBP2, MCL-1, phospho-S6 (S240/244) (Cell Signaling Technology, Beverly, MA), actin (Sigma-Aldrich). Antibody dilutions were performed according to the manufacturer's instructions. Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare Life sciences, Pittsburgh, PA) after incubation with HRP conjugated secondary antibody (Promega, Madison, WI).

Mallya S., Fitch B.A., Lee J.S., So L., Janes M.R, & Fruman D.A. (2014). Resistance to mTOR Kinase Inhibitors in Lymphoma Cells Lacking 4EBP1. PLoS ONE, 9(2), e88865.

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eIF4E Protein Complex Isolation

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Samples (1×10⁷ cells) were lysed in 500μl of RIPA lysis buffer and clarified by centrifugation (13,000g, 20 minutes, 4°C). Aliquots of supernatants were incubated with 50μl of 7^m-GTP-Sepharose beads (GE Healthcare) for 1 to 2 hours at 4°C. The beads were then washed 3 times in 1ml of phosphate-buffered saline (PBS) and boiled in 50μl of SDS-PAGE sample buffer for 7 minutes. The retained supernatants were analyzed by Western blot with primary antibodies against total 4EBP1, eIF4E, and eIF4G (Cell Signaling Technology, USA), as described in section 5 of the methods section.

Shi F., Len Y., Gong Y., Shi R., Yang X., Naren D, & Yan T. (2015). Ribavirin Inhibits the Activity of mTOR/eIF4E, ERK/Mnk1/eIF4E Signaling Pathway and Synergizes with Tyrosine Kinase Inhibitor Imatinib to Impair Bcr-Abl Mediated Proliferation and Apoptosis in Ph+ Leukemia. PLoS ONE, 10(8), e0136746.

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Antibody Protocol for Protein Detection

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The following antibodies were purchased from Cell Signaling (Leiden, Netherlands): S6 (#2317), pS6 (#4858), actin (#4967), eIF3A (#3411), eIF4G (#2498), RPL5 (#51345) and GAPDH (#2118). FLAG-HRP (A8592) antibody was purchased from Sigma; RPLP0 (ab192866), RPS3 (#128995), and APP (ab2071) from Abcam (Cambridge, UK), and anti-beta-amyloid 6E10 from BioLegend (803001).

Matthes F., Hettich M.M., Schilling J., Flores-Dominguez D., Blank N., Wiglenda T., Buntru A., Wolf H., Weber S., Vorberg I., Dagane A., Dittmar G., Wanker E., Ehninger D, & Krauss S. (2018). Inhibition of the MID1 protein complex: a novel approach targeting APP protein synthesis. Cell Death Discovery, 4, 4.

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