Phospho stat2 tyr690

Manufactured by Cell Signaling Technology

The Phospho-STAT2 (Tyr690) product is an antibody that specifically recognizes the phosphorylated form of the STAT2 protein at the Tyr690 residue. This antibody can be used to detect and quantify the phosphorylation status of STAT2 in various experimental settings.

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Phospho-STAT2 STAT2

3 protocols using phospho stat2 tyr690

Immunophenotyping of Immune Cells

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Zoledronic acid, LPS (lipopolysaccharide), puromycin, and G418 were obtained from Sigma‐Aldrich. Antibodies against human HLA‐G (sc‐21799) and β‐actin (sc‐47778) were purchased from Santa Cruz Biotechnology. PD‐L1 antibody (17952‐1‐AP) and PD‐L1 (22C3) were purchased from Proteintech and Agilent Technologies, respectively. Antibodies specific for CD3ε (NB600‐1441), PD‐L1 (FAB1561R, FAB1561G), CD4 (FAB3791R), and CD8 (NBP2‐34590AF488) were purchased from Novus Biologicals. HLA‐G (PE: #335906, Alexa Fluor 488: #335918), PD‐1 (#367412), CD3 (#317318), CD8 (#344704), CD56 (#304611), CD66b (#305104), and Vδ2 (#331418) were purchased from BioLegend. Anti‐Tyk2 (phospho‐Tyr1054/1055) (orb505746) antibody was obtained from Biorbyt. Antibodies specific for phospho‐ZAP70/Syk (Tyr319, Tyr352) (#MA5‐36963) and HLA‐G (MEM‐G/2: #MA1‐19394, 87G: MA1‐10356) were purchased from Thermo Fisher Scientific. Antibodies for CD14 (#12‐0149‐42), TCRγδ (#12‐9959‐42), TCRαβ (#11‐9955‐42), and NKG2D (#12‐5878‐42) were purchased from eBioscience. Vγ9 (#555732), hMito (ab92824), and VHH (iFluor 647: A02019, HRP: A2016) were purchased from BD Pharmingen, Abcam, and GenScript, respectively. Phospho‐Stat2 (Tyr690) (#77366) and HLA‐G (#79769) were obtained from Cell Signaling Technology. Recombinant human interleukin 2 (IL‐2) was obtained from Thermo Fisher Scientific.

Huang S., Pan C., Lin Y., Chen M., Chen Y., Jan C., Wu C., Lin F., Wang S., Lin C., Lin P., Huang W., Chiang Y., Tsai W., Chiu Y., Lin T., Chiu S, & Cho D. (2023). BiTE‐Secreting CAR‐γδT as a Dual Targeting Strategy for the Treatment of Solid Tumors. Advanced Science, 10(17), 2206856.

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Immunoblotting of Phosphorylated Proteins

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For immunoblotting, murine CD4⁺ T cells were isolated from mouse spleen and peripheral lymph node single cell suspension, and human CD4⁺ T cells were enriched from healthy donor PBMC with STEMCELL mouse or human CD4⁺ T cell isolation kits, respectively. Cells were lysed in lysis buffer with protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentration in samples was quantified by BCA assay (Thermo Fisher Scientific) before loading the samples for electrophoresis and membrane transfer. The transferred membrane was blocked with TBST (0.1% Tween 20) containing 5% BSA for 1 h at room temperature. The membrane was incubated with primary antibodies overnight including anti-phospho-AKT (Ser473) (clone: D9E; Cell Signaling), AKT (pan) (Clone: 40D4; Cell Signaling), phospho-STAT1 (Tyr701) (clone: D4A7; Cell Signaling), phospho-STAT2 (Tyr690) (Cell Signaling), phospho-S6RP (Ser235/236) (clone: D57.2.2E; Cell Signaling), phospho-p70 S6 Kinase (Thr389) (Cell Signaling), phosphor-4EBP1 (Thr37/46) (clone: 236B4; Cell Signaling), anti-RICTOR (Clone: 53A2; Cell Signaling) and anti-β-actin (clone: 13E5; Sigma-Aldrich). Then, the membrane was washed and incubated with the corresponding secondary antibody for subsequent enhanced chemiluminescence (ECL; Thermo Fisher) exposure. The band intensity of all the immunoblot was analyzed by ImageJ software.

Zhou X., Qi H., Li M., Li Y., Zhu X., Amin S., Alexander M., Diadhiou C., Davidson A, & Zeng H. (2022). mTORC2 contributes to systemic autoimmunity. Immunology, 168(3), 554-568.

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Immunoblotting for Phosphoprotein Detection

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For immunoblotting, cells were lysed in lysis buffer with protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentration in samples was quantified by BCA assay (Thermo Fisher Scientific) before loading the samples for electrophoresis and membrane transfer. The transferred membrane was blocked with TBST (0.1% Tween 20) containing 5% BSA for 1 h at room temperature. The membrane was incubated with primary antibodies overnight including antiphospho-AKT (Ser473) (clone: D9E; Cell Signaling), phospho-STAT1 (Tyr701) (clone: D4A7; Cell Signaling), phospho-STAT2 (Tyr690) (Cell Signaling), phospho-S6 (Ser235/236) (clone: D57.2.2E; Cell Signaling) and anti-β-actin (clone: 13E5; Sigma-Aldrich). Then, the membrane was washed and incubated with the corresponding secondary antibody for subsequent enhanced chemiluminescence (ECL; Thermo Fisher) exposure. The band intensity of all the immunoblot was analyzed by ImageJ software.

Zhou X., Qi H., Li M., Li Y., Zhu X., Amin S., Alexander M.P., Diadhiou C.M., Davidson A., & Zeng H. (2021). mTORC2 contributes to murine systemic autoimmunity.

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