T cells were lysed in Ripa buffer containing a protease inhibitor and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates were centrifuged at 4 °C at 12,000× g for 10 min, separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked with 5% BSA in PBS containing 0.05% Tween-20 and incubated with various antibodies recognizing pAMPKα1 (T172), pAMPKα1 (S485), pS6 (S235/236), peIF4E (S209), pULK1 (S555), ATG7, AQP9, PGC1α, TFAM, OPA1, pDRP1 (S616), ID2, ID3, cMyb, cMyc, Blimp1, T-bet, ZEB2, FOXO1, TCF1, TRAF6, pTSC2 (S1387), Eomes, HIF-1α and β-actin (Cell Signaling Technology) and the Complex I subunit NDUFA9 (Abcam, Cambridge, MA, USA). The membranes were imaged using a BioRad Chemidoc MP (Bio-Rad, Hercules, CA, USA) after a second incubation step with a horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Cell Signaling) secondary antibody [18 (link)].
Eomes
Manufactured by Cell Signaling Technology
Sourced in United States
Eomes is a lab equipment product manufactured by Cell Signaling Technology. It is a protein that plays a critical role in cellular processes. The core function of Eomes is to regulate gene expression and cell differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Ndufa9, by Abcam (1 mentions)
P tsc2, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated goat antirabbit igg or goat antimouse igg, by Cell Signaling Technology (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Facsaria, by BD (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Facsaria fusion, by BD (1 mentions)
Bromodeoxyuridine (brdu), by BD (1 mentions)
Cd69 h1.2f3, by BioLegend (1 mentions)
Cx3cr1, by BioLegend (1 mentions)
Tcf1 c63d9, by Cell Signaling Technology (1 mentions)
Cd45.2 104, by BioLegend (1 mentions)
2 protocols using eomes
1
Western Blot Analysis of Cellular Signaling
2
Isolation and Staining of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Single-cell suspensions were prepared from spleen or lymph node by mechanical disruption, and intestinal tissue and tumor were processed as described previously (Milner et al., 2017 (link)). RBCs were lysed with ACK buffer (140 mM NH4Cl and 17 mM Tris-base, pH 7.4). The following antibodies were used for surface staining (all from eBioscience unless otherwise specified): CD8 (53–6.7), CD27 (LG-7F9), CD43 (eBioR2.60), CD44 (IM7), CD45.1 (A20-1.7), CD45.2 (104), CD69 (H1.2F3; BioLegend), CD103 (2E7), Tim3 (RMT3-23), CD127 (A7R34), CD62L (MEL-14), KLRG1 (2F1), PD-1 (J43), and CX3CR1 (SA011F11; BioLegend); or intracellular staining: GzB (GB12; Invitrogen), Eomes (Dan11mag), TCF1 (C63D9; Cell Signaling Technology), T-bet (4B10), and Ki-67 (SolA15). The H-2Db-GP33-41 tetramer was obtained from the National Institutes of Health Tetramer Core. Intracellular staining was performed using the Foxp3 Transcription Factor Staining Buffer Set (eBioscience). CellTrace Violet (Ebioscience) and BrdU (BD PharMingen) were used per manufacturer instructions where 2 mg BrdU was administered i.p. 2 h before tissue harvesting. For flow cytometry analysis, all data were acquired on a BD LSRFortessa X-20 or a BD LSRFortessa. Cell sorting was performed on BD FACSAria or BD FACSAria Fusion instruments.
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