The anti-Fas (IgM) antibody, anti-phospho-LAT-Tyr226, and rabbit polyclonal anti-human LAT were from Merck-Millipore; anti-PLC-γ1 mAb, anti-Lck, and anti-Erk were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies binding phospho-Erk, phospho-PLC-γ1-Tyr783, phospho-ZAP70-Tyr319, phospho-MEK-Ser221, phospho-LAT-Tyr191, and anti-MEK were from Cell Signaling Technology; anti-6His-HRP, anti-Grb2, and anti-phospho-LAT-Tyr132 antibodies were from Abcam (Cambridge, MA, USA); and anti-CD69-APC (allophycocyanin) and anti-β-actin monoclonal antibodies were from Biolegend. The protein synthesis inhibitor cicloheximide was purchased from Merck-Millipore. Stimulations were performed with anti-human CD3 (OKT3; eBioscience) or anti-human CD28 (CD28.2; BD Pharmingen) monoclonal antibodies.
Phospho zap70 tyr319
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-ZAP70-Tyr319 is a laboratory reagent used to detect the phosphorylation of the tyrosine 319 residue of the ZAP70 protein. ZAP70 is a key signaling molecule involved in T-cell activation.
Automatically generated - may contain errors
Lab products found in correlation
Phospho plc γ1 tyr783, by Cell Signaling Technology (2 mentions)
Phospho mek ser221, by Cell Signaling Technology (2 mentions)
Anti mek, by Cell Signaling Technology (2 mentions)
Anti lck, by Santa Cruz Biotechnology (2 mentions)
Zap70, by Cell Signaling Technology (2 mentions)
Anti lat, by Santa Cruz Biotechnology (2 mentions)
Anti erk, by Santa Cruz Biotechnology (1 mentions)
Anti fas igm antibody, by Merck Group (1 mentions)
Anti phospho lat tyr132, by Abcam (1 mentions)
Anti human cd3 okt3, by Thermo Fisher Scientific (1 mentions)
Anti human cd28 cd28.2, by BD (1 mentions)
Anti 6 his hrp, by Abcam (1 mentions)
Phospho lat tyr191, by Cell Signaling Technology (1 mentions)
Anti grb2, by Abcam (1 mentions)
Anti erk antibody, by Santa Cruz Biotechnology (1 mentions)
Anti cd3 okt3, by BioLegend (1 mentions)
Anti plc γ1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by BioLegend (1 mentions)
Phospho cas l, by Cell Signaling Technology (1 mentions)
Zap70 clone 99f2, by Cell Signaling Technology (1 mentions)
5 protocols using phospho zap70 tyr319
1
T Cell Activation Signaling Pathway
2
Antibody Panel for Signal Transduction
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Anti-LAT, anti-PLC-γ1, and anti-Erk antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-NTAL NAP-07 monoclonal antibody was from EXBIO (Prague, Czech Republic). Antibodies binding phospho-Erk, phospho-PLC-γ1-Tyr783, ZAP70, phospho-ZAP70-Tyr319, phospho-MEK-Ser221, and anti-MEK were from Cell Signaling Technology. Anti-β-actin, anti-CD3 (OKT3), and anti-CD4 monoclonal antibodies were provided by Biolegend (San Diego, CA, USA).
3
Immunofluorescence Analysis of Signaling Proteins
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After incubating cells for 3 min at 37 °C, cells were fixed with 2% paraformaldehyde, and permeabilized with 0.01% Triton‐X in phosphate‐buffered saline for 2 min, followed by blocking with 5% casein for 30 min. Primary antibody incubation was performed overnight at 4 °C, followed by fluorescently labeled secondary antibody incubation (with or without Phalloidin) for 1 h at room temperature. Primary antibodies used: Cas‐L (clone 2G9; Novus Biologicals, Littleton, CO, USA, #NB100‐1699); phospho‐Cas‐L (Cell Signaling, Danvers, MA, USA, #4015); ZAP70 (clone 99F2; Cell Signaling #2705); phospho‐ZAP70 (Tyr319; Cell Signaling #2701). Fluorescently labeled secondary antibodies were obtained from Molecular Probes.
4
T-cell Activation Signaling Pathway
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Phorbol 12-myristate 13-acetate (PMA) and ionomycin (were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were prepared in DMSO. Anti-LAT, anti-Lck, anti-PLC-γ, and phospho-Erk antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany); antibodies binding ZAP-70, phospho-ZAP-70-Tyr319, β-actin, phospho-PLC-γ1-Tyr783, and phospho-LAT-Tyr171 were from Cell Signaling Technology (Danvers, MA, USA); anti-Grb2 mAb was from Abcam (Cambridge, MA, USA); and anti-Erk mAb was from BioLegend (San Diego, CA, USA). Stimulations were performed with the anti-human CD3 OKT3 monoclonal antibody (eBioscience, Thermo Fisher Scientific). Staining to assess CD3 expression on the membrane was performed with OKT3 antibody conjugated to APC (BioLegend, San Diego, CA, USA).
5
Visualizing Jurkat T Cell Cytoskeleton
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WT, YFP-ZAP-70, or GFP-centrin Jurkat T cells were fixed 6 min after being added to the substrates using 4% PFA (Electron Microscopy Sciences). Cells were then permeabilized using 0.15% TritonX and the samples were blocked using 0.3 M glycine and bovine serum albumin. For MT staining, beta tubulin antibody (Thermofisher Cat#PA5-16863) was used as primary antibody and Alexa Fluor-488 (Invitrogen, Cat# A10235) was used for secondary antibody. For pZap70 staining phospho-Zap70 Tyr319 (Cell Signaling Cat# 2701) primary antibody was used. After labeling of MTs or pZap70, F-actin was stained using rhodamine phalloidin (Cytoskeleton, Cat#PHDR1).
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