Briefly, the mice organs were collected and disrupted in lysis buffer containing protease inhibitors (Roche Diagnostics, Berlin, Germany). After centrifugation, the supernatant was collected, and equivalent amounts of protein were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The protein bands were visualized using DyLight 800/DyLight 680-conjugated secondary antibodies, and an infrared fluorescence image was obtained using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Western blot analyses were performed by ImageJ with anti-Gapdh (KM9002, Sungene, Tianjin, China), anti-Lc3b (SAB4200361, Sigma, St Louis, MO, USA), anti-Sqstm1 (PM045, MBL International, Japan), and anti-Eva1a (NB110-74787, Novusbio, Littleton, CO, USA) antibodies. Antibodies against Ulk1, Akt, Mtor, Erk1/2, Lkb1, Ampk, Rps6kb1, and Eif4ebp1 and against phosphorylated Ulk1, Akt, Mtor, Erk1/2, Lkb1, Ampk, Rps6kb1, and Eif4ebp1 were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against Drp1, Tomm20, Pink1, Parkin, Bnip3, Mitofusin2, and Pgc1 were purchased from Abcam (Cambridge, UK). DyLight 800/DyLight 680-conjugated secondary antibodies against mouse or rabbit IgG were purchased from Rockland Immunochemicals (Limerick, PA, USA).
Eif4ebp1
Manufactured by Cell Signaling Technology
Sourced in United States
EIF4EBP1 is a protein that functions as a repressor of the eukaryotic translation initiation factor eIF4E. It plays a role in the regulation of protein synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Phospho eif4ebp1, by Cell Signaling Technology (3 mentions)
Goat anti mouse and goat anti rabbit fluorescence dye labelled antibodies, by LI COR (2 mentions)
Maxisorp microtitre plate, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
P70s6k, by Cell Signaling Technology (2 mentions)
Rps6kb1, by Cell Signaling Technology (1 mentions)
Tomm20, by Abcam (1 mentions)
Bnip3, by Abcam (1 mentions)
Anti lc3b sab4200361, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Mitofusin 2, by Abcam (1 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
Rabbit anti mouse secondary antibody, by Abcam (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Mouse monoclonal antibodies, by Abcam (1 mentions)
Phospho ulk1, by Cell Signaling Technology (1 mentions)
Phospho akt ser, by Cell Signaling Technology (1 mentions)
G6pdh, by Merck Group (1 mentions)
10 protocols using eif4ebp1
1
Western Blot Analysis of Autophagy and Mitochondrial Markers
2
Immunosuppressive Factors in Cancer
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RPMI-1640 medium, foetal bovine serum, supplements as well as basic laboratory chemicals were purchased from Sigma (Suffolk, UK). Maxisorp™ microtitre plates were obtained from Nunc (Roskilde, Denmark) and Oxley Hughes Ltd (London, UK). Mouse monoclonal antibodies directed against HIF-1α, mTOR and β-actin, as well as rabbit polyclonal Antibodies against phospho-S2448 mTOR, RAGE and HRP-labelled rabbit anti-mouse secondary antibody were purchased from Abcam (Cambridge, UK). Antibodies against phospho-S65 and non-phosphorylated (total) eukaryotic initiation factor 4E binding protein 1 (eIF4E-BP1) were obtained from Cell Signaling Technology (Danvers, MA USA). Goat anti-mouse and goat anti-rabbit fluorescence dye-labelled antibodies were obtained from LI-COR (Lincoln, Nebraska USA). ELISA-based assay kits for the detection of TNFα, IL-1β, SCF and VEGF were purchased from Bio-Techne (R&D Systems, Abingdon, UK). Anti-Tim-3 mouse monoclonal antibody, its single chain variant as well as human Ig-like V-type domain of Tim-3 (amino acid residues 22–124) and human HMGB1 expressed and purified from E. coli (see below for more details) were used in our experiments.11 (link),15 (link) All other chemicals purchased were of the highest grade of purity commercially available.
3
Western Blot Antibody Panel for Cell Signaling
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Primary antibodies against GFP (Takara Bio, 632380, AB_10013427; 1:5000), G-6-PDH (Sigma-Aldrich, A9521, AB_258454; 1:5000), Adh1 (Millipore, 126745, AB_564196; 1:200000), RPS6 (Cell Signaling Technology, 2217, AB_331355; 1:1000), phospho-RPS6 (Ser235-236) (Cell Signaling Technology, 4856, AB_2181037; 1:1000), RPS6KB (Cell Signaling Technology, 2708, AB_390722; 1:1000), phospho-RPS6KB (Thr389) (Cell Signaling Technology, 9205, AB_330944; 1:1000), EIF4EBP1 (Cell Signaling Technology, 9452, AB_331692; 1:1000), phospho-EIF4EBP1 (Thr37/46) (Cell Signaling Technology, 2855, AB_560835; 1:1000), phospho-EIF4EBP1 (Ser65) (Cell Signaling Technology, 9451, AB_330947; 1:1000), AKT1 (Cell Signaling Technology, 4691, AB_915783; 1:1000), phospho-AKT(Ser473) (Cell Signaling Technology, 4060, AB_2315049; 1:1000), phospho-AKT(Thr308) (Cell Signaling Technology, 13038, AB_2629447; 1:1000), ULK1 (Cell Signaling Technology, 8359, AB_11178668; 1:1000), phospho-ULK1(Ser757) (Cell Signaling Technology, 6888, AB_10829226; 1:1000), MAP1LC3 AB (Cell Signaling Technology, 12741, AB_2617131; 1:1000), β-actin (Cell Signaling Technology, 4967, AB_330288; 1:1000) were used.
4
Western blot analysis of HCC samples
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Of the 40 paired HCC samples, randomly selected 8 HCCs and matched NCL tissues were homogenized in a RIPA lysis buffer. After centrifugation at 12,000 rpm and 4°C for 20 minutes, approximate 40 μg of protein were run on a 12% SDS-PAGE gel and transferred to polyvinylidene difluoride membrane (PVDF, Millipore). After blocking non-specific binding sites for 60 minutes with 5% non-fat milk, membranes were incubated with rabbit monoclonal antibody against EIF4EBP1 (1:1000; Cell signaling Technology), and GAPDH (1:1000; Sigma-Aldrich) at 4°C over night, respectively. Then membranes were washed three times with TBST for 15 minutes each time and incubated with HRP-conjugated anti-rabbit secondary antibody (1:1000; Santa Cruz) for 45 minutes at room temperature. The membrane was developed by an enhanced chemiluminescence system (ECL; cell signaling) after washed three times with TBST. The intensity of the protein bands was determined by using Image J software (http://rsb.info.nih.gov/ ).
5
Western Blot and Immunofluorescence Antibodies
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The following antibodies were used for Western blotting and immunofluorescence: YB-1 (Abcam: Ab12148, Proteintech Group: 20339-1-AP, Bethyl Laboratories, Inc (A): A404-230-T, Bethyl Laboratories, Inc (B): A303-231-T), Dach1 (Proteintech Group: 10914-1-AP), Caprin1 (Proteintech Group: 14112-1-AP), G3BP1 (Santa Cruz Biotechnology, Inc: sc-81940), G3BP2 (Bethyl Laboratories, Inc: A302-040A), TIA-1 (Santa Cruz Biotechnology, Inc: sc-1751), TIAR (Santa Cruz Biotechnology, Inc: sc-1749), eIF3b (Santa Cruz Biotechnology, Inc: sc-16377), eIF4G (Santa Cruz Biotechnology, Inc: sc-11373), eIF4E-BP1 (Cell Signaling Technology: 9452S), PABP (Santa Cruz Biotechnology, Inc: sc-32318), Nucleolin (Santa Cruz Biotechnology, Inc: sc-9893), eIF4E (Santa Cruz Biotechnology, Inc: sc-9976), HuR (Santa Cruz Biotechnology, Inc: sc-5261), Fxr2 (Santa Cruz Biotechnology, Inc: sc32266), Rack1 (Santa Cruz Biotechnology, Inc: sc-17754), RPS23 (Santa Cruz Biotechnology, Inc: sc-100837), Twist1 (Bethyl Laboratories, Inc: A301-394A), Snail1 (Origene: TA500416), Zeb1 (Bethyl Laboratories, Inc: A301-921A), Puromycin (EMD Milipore: 12D10), GFP (Applied Biological Materials: G160), β-actin (Proteintech Group: 66009-1).
6
Protein Expression Analysis in Skeletal Muscle
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Total protein was extracted from skeletal muscle by the protein extraction Kit (KeyGEN). Protein concentration was determined with BCA Protein Assay Kit (Takara), and an equal amount of protein was separated on SDS-polyacrylamide gel electrophoresis. After transferring to a polyvinylidene fluoride (PVDF) membrane (Millipore), the primary antibody and the corresponding secondary antibody were used to detect protein of interest. The antibody used in this study were as follows: Atrogin-1 (Abclonal); MuRF-1 (Abclonal); LC3 (Abclonal); P62 (Abclonal); Ndufb2 (Abclonal); Clusterin (Abclonal); IGF1 (Abclonal); PI3K(p85α) (Abclonal); P-mTOR (Abclonal); mTOR (Abclonal); P-P70S6K (Abclonal); P70S6K (Abclonal); P-FOXO3A (Abclonal); FOXO3A (Abclonal); P-EIF4EBP1 (Abclonal); EIF4EBP1 (Abclonal); P-AKT (Cell Signaling Technology); AKT (Proteintech); and GAPDH (Bioworld Technology). The protein was visualized using High-sig ECL Western Blotting Substrate (Tanon) and imaged using the Tanon-5200S Chemiluminescent Imaging System (Tanon).
7
Antibodies and Reagents for mTOR Signaling
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Antibodies against HMGA1 (ab129153), HMGA1-ChIP Grade (ab252930), and FKBP12 (ab2918) were purchased from Abcam. Antibodies against HMGA1 (sc-393213) and FKBP12 (sc-133067) and mouse IgG (sc-2025) were purchased from Santa Cruz Biotechnology. Antibodies against S6 ribosomal protein (#64108) and phospho-S6 ribosomal protein (#81736) and rabbit IgG (#2729), mTOR (#2983), phospho-mTOR (#5536), eIF4EBP1(#9452), and phospho-eIF4EBP1(#9456) were purchased from Cell Signaling Technology (Danvers, MA). Antibody against beta actin (81115-1-RR) was purchased from Protein Tech (Wuhan, China). Rapamycin was obtained from MedChemExpress (HY-10219).
8
Investigating HIF-1α and mTOR Signaling
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RPMI-1640 medium, foetal calf serum and supplements, P. aeruginosa LPS, were purchased from Sigma (Suffolk, UK). Maxisorp™ microtitre plates were obtained either from Nunc (Roskilde, Denmark) or kindly provided by Oxley Hughes Ltd (London, UK). Mouse monoclonal antibodies to HIF-1α, mTOR and β-actin as well as rabbit polyclonal antibodies against phospho-S2448 mTOR were purchased from Abcam (Cambridge, UK). Antibodies against phospho-T389 p70 S6 kinase 1 (p70 S6K1), total and phospho-S65 eukaryotic initiation factor 4E binding protein 1 (eIF4E-BP1) antibodies were obtained from Cell Signaling Technology (Danvers, MA USA). Goat anti-mouse and goat anti-rabbit fluorescence dye-labelled antibodies were obtained from Li-Cor (Lincoln, Nebraska USA). ELISA-based assay kits for the detection of VEGF, TNF-α and IL-6 were purchased from Bio-Techne (R&D Systems, Abingdon, UK). All other chemicals were of the highest grade of purity and available commercially unless otherwise stated.
9
Comprehensive Protein Expression Analysis
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TA muscle samples and C2C12 cells were homogenized and lysed in radioimmunoprecipitation assay (RIPA) buffer with complete mini protease/phosphatase inhibitor cocktail (5872S, Cell signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. Western blot analysis was performed according to our previous protocol [15 (link)]. Primary antibodies used were GAPDH (1:2000, MA5-15738, Invitrogen, Waltham, MA, USA), PI3 Kinase p85 (1:2000, 4292, Cell signaling Technology, Danvers, MA, USA), Akt (1:2000, 4691, Cell signaling Technology, Danvers, MA, USA), pAkt (1:2000, 4060, Cell signaling Technology, Danvers, MA, USA), mTOR (1:2000, 5043, Cell signaling Technology, Danvers, MA, USA), p70-S6K (1:2000, 34475, Cell signaling Technology, Danvers, MA, USA), EIF4EBP1 (1:2000, 9644, Cell signaling Technology, Danvers, MA, USA) and MyoD (1:2000, 13812, Cell signaling Technology, Danvers, MA, USA). Secondary antibodies used were anti-rabbit IgG, HRP-linked antibody (1:5000, Cell signaling Technology, Danvers, MA, USA) and goat anti-mouse IgG (H + L) antibody (1:5000, Invitrogen, Waltham, MA, USA). Relative protein contents were imaged by GeneGnome XRQ (Syngene, Cambridge, UK) and quantified by ImageJ.
10
Western Blot Analysis of Zebrafish Ganglioneuroma Cells
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Protein was extracted from zebrafish primary ganglioneuroma cells using RIPA buffer after compound treatment for 18 h. Western blotting was performed as described previously (Tao et al., 2013 (link)). Protein samples were separated by a 10% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (#PI88518; Thermo Fisher Scientific). The membrane was blocked in PBST containing 5% nonfat milk for 1 h at room temperature, incubated with a primary antibody for 2 h diluted in blocking buffer. After washing three times in PBST, the membrane was incubated with a secondary antibody for 1 h diluted in blocking buffer and washed three times again in PBST. The results were visualized on autoradiography films with SuperSignal West Pico Chemiluminescent Substrate (#PI34080; Pierce) or SuperSignal West Dura Extended Duration Substrate (#34075; Pierce). Antibodies against phospho-S6 ribosomal protein (Ser235/236, #2211; Cell Signaling Technology), S6 ribosomal protein (#2317; Cell Signaling Technology), phospho-EIF4EBP1 (Thr37/46, #2855; Cell Signaling Technology), EIF4EBP1 (#9644; Cell Signaling Technology), and α-Tubulin (#T6074; Sigma) were used as primary antibodies.
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