Spheroid and colon tissue samples were collected and flash frozen. After 24 hours, protein was extracted and immunoblotting performed as previously described (50). The membranes were blocked with 5% non-fat dry milk for one hour and then probed with primary antibodies against phospho RPS6 (Ser235/236, #4858, Cell Signaling Technology), phospho AKT (Ser473, #4060, Cell Signaling Technology), phospho 4EBP1 (Thr37/46, #2855, Cell Signaling Technology), phospho EEF2 (Thr 56, #2331, Cell Signaling Technology), total RPS6 (#2217, Cell Signaling Technology), total AKT (#4691, Cell Signaling Technology), total 4EBP1 (9644, Cell Signaling Technology)), or total eEF2 (#2332, Cell Signaling Technology) in bovine serum albumin at 1:1000. Anti-GAPDH (#8884, Cell Signaling Technology) or β-actin (#5125, Cell Signaling Technology) antibodies were used as a loading control at a ratio of 1:1000.
Phospho eef2
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-eEF2 is a laboratory product that detects the phosphorylation state of the eukaryotic elongation factor 2 (eEF2) protein. eEF2 is a key regulator of protein translation in cells. The Phospho-eEF2 product allows for the measurement of eEF2 phosphorylation levels, which can provide insights into cellular signaling pathways and translational control mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Total eef2, by Cell Signaling Technology (2 mentions)
Phospho 4ebp1, by Cell Signaling Technology (2 mentions)
Total 4e bp1, by Cell Signaling Technology (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Phospho rps6, by Cell Signaling Technology (1 mentions)
Total rps6, by Cell Signaling Technology (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Psd95, by Cell Signaling Technology (1 mentions)
Total akt, by BD (1 mentions)
T per reagent, by Thermo Fisher Scientific (1 mentions)
Phospho trkb, by Abcam (1 mentions)
Phospho p70s6k, by Cell Signaling Technology (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
β actin, by Abcam (1 mentions)
Total ampkα, by Cell Signaling Technology (1 mentions)
Phospho ulk1, by Cell Signaling Technology (1 mentions)
Phospho ampkα, by Cell Signaling Technology (1 mentions)
Total ulk1, by Cell Signaling Technology (1 mentions)
4 protocols using phospho eef2
1
Immunoblotting Analysis of Signaling Pathways
2
Profiling Signaling Pathways in Brain Tissue
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Brain tissues were homogenized in 20mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid containing 0.32M sucrose and centrifuged at 700 g for 10 minutes at 4°C. The supernatants were centrifuged again at 14,000 g for 10 minutes at 4°C, and the pellets were resuspended in T-PER reagent (Thermo Scientific, Rockford, IL). Proteins were separated and transferred onto a nitrocellulose membrane. Blots were immunostained overnight at 4°C with primary antibody against total GSK-3β (BD, Franklin Lakes, NJ), phospho-GSK-3β at Ser9, total Akt (the serine/threonine kinase, also known as protein kinase B or PKB), phospho-Akt at Ser473, total extracellular signal-regulated kinases (ERKs), phospho-ERK at Thr202/Tyr204, total mTOR, phospho-mTOR at Ser2448, total P70S6 kinase (P70S6K), phospho-P70S6K at Thr389, total eukaryotic elongation factor-2 (eEF2), phospho-eEF2 at Thr56, PSD95 (all from Cell Signaling, Beverly, MA), total tropomyosin-related kinase B (TrkB; Millipore, Billerica, MA), phospho-TrkB at Tyr817, or the house-keeping gene β-actin (Abcam, Cambridge, MA). Membranes were then incubated with secondary antibodies (LI-COR, Lincoln, NE) for 1 hour at room temperature. Finally, blotted proteins were detected and quantified using the Odyssey infrared imaging system (LI-COR).
3
Molecular Signaling Pathway Analysis
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DL-HICA was obtained from Sigma-Aldrich (St. Louis, MO, USA). HICA was dissolved in Dulbecco’s modified Eagle’s medium (DMEM) at a concentration of 100 mM, and the solution was neutralized with NaOH and filtered through a 0.22-µm pore polyvinylidene fluoride (PVDF) membrane; this neutralized HICA solution was used to prepare relevant medium samples. Mouse recombinant TNFα and IFNγ were obtained from R&D systems (Minneapolis, MN, USA). Reagents not specifically mentioned were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) or Sigma-Aldrich.
The following antibodies were obtained from Cell Signaling Technology (CST, Beverly, MA, USA): phospho-AMPKα, total-AMPKα, phospho-ACC, total-ACC, phospho-ULK1, total-ULK1, total-p70S6K, phosopho-4E-BP1, total-4E-BP1, phospho-ERK1/2, total-ERK1/2, phospho-p38MAPK, total-p38MAPK, phospho-eEF2, total-eEF2, phospho-STAT3, total-STAT3, phospho-p65NFκB, total NF-κB, and β-tubulin. Phospho-p70S6K, iNOS, and GAPDH antibodies were obtained from Affinity Biosciences (Cincinnati, OH, USA), BD Biosciences (Franklin Lakes, NJ, USA), and GenTex (Irvine, CA, USA), respectively. Atrogin-1 and muscle RING-finger protein-1 (MURF-1) antibodies were obtained from Abcam (Cambridge, UK).
The following antibodies were obtained from Cell Signaling Technology (CST, Beverly, MA, USA): phospho-AMPKα, total-AMPKα, phospho-ACC, total-ACC, phospho-ULK1, total-ULK1, total-p70S6K, phosopho-4E-BP1, total-4E-BP1, phospho-ERK1/2, total-ERK1/2, phospho-p38MAPK, total-p38MAPK, phospho-eEF2, total-eEF2, phospho-STAT3, total-STAT3, phospho-p65NFκB, total NF-κB, and β-tubulin. Phospho-p70S6K, iNOS, and GAPDH antibodies were obtained from Affinity Biosciences (Cincinnati, OH, USA), BD Biosciences (Franklin Lakes, NJ, USA), and GenTex (Irvine, CA, USA), respectively. Atrogin-1 and muscle RING-finger protein-1 (MURF-1) antibodies were obtained from Abcam (Cambridge, UK).
4
Western Blot Analysis of Cardiac Proteins
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