Anti pzap70

Manufactured by Cell Signaling Technology

Anti-pZAP70 is a monoclonal antibody that recognizes phosphorylated ZAP70. ZAP70 is a protein tyrosine kinase that plays a critical role in T-cell receptor (TCR) signaling. Phosphorylation of ZAP70 is an important event in the activation of T-cells.

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3 protocols using anti pzap70

Detailed Immunological Antibody Protocols

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The antibodies used in this study for flow cytometry include: anti-mouse CD8 (#563786), anti-mouse CD25 (#558642), anti-mouse TCRβ (#553174), anti-human CD3 (#563798), anti-human CD8 (#563795), and anti-human TCRαβ (#563826) from BD Biosciences; anti-mouse CD4 (#100544), anti-mouse IFN-γ (#505835), anti-human LFA-1 (#363404), anti-human CD11a (#301208), anti-human CD18 (#302114), anti-human CD25 (#302625), and anti-human CD69 (#310920) from BioLegend; anti-mouse CD4 (#17-0042-83), anti-mouse CD4 (#17-0041-83), anti-mouse CD69 (#25-0691-82), anti-mouse TNF (#17-7321-82), anti-mouse IL-2 (#12-7021-82), anti-human CD4 (#17-0048-2), and anti-human CD8 (#11-0088-41) from eBioscience.
The antibodies used for immunoprecipitation and immunoblotting include: anti-Cdc7 (#ab10535), anti-Dbf4 (#ab124707), anti-RNA polymerase II (#ab817) and anti-RNA polymerase II (phospho S2, # ab193468) from Abcam; anti-PLCγ (#610027) and anti-ERK (#610031) from BD Biosciences; anti-LAT (#641102) from BioLegend; anti-GAPDH (#2118), anti-pPLCγ (#2821), anti-ZAP70 (#2709), anti-pZAP70 (#2717), anti-pERK (#4370), anti-pLAT (#3584), anti-pLck (#6943), and anti-NF-κB (#3035) from Cell Signaling; anti pMCM2 S40 (#GTX62847) from GeneTex; anti-MCM2 (#MA5-15895) from Pierce; and anti-Lck (#sc-433) from Santa Cruz.

Chen E.W., Tay N.Q., Brzostek J., Gascoigne N.R, & Rybakin V. (2019). A Dual Inhibitor of Cdc7/Cdk9 Potently Suppresses T Cell Activation. Frontiers in Immunology, 10, 1718.

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Immunoblot Analysis of Cell Signaling

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Sorted cells or stimulated cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 x Roche Complete Protease Inhibitor Cocktail, and 1 x Roche Phosphatase Inhibitor Cocktail). Protein concentrations were measured using a BCA kit (Pierce). Equal amounts of protein samples were separated by 9% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad). The following primary antibodies were used: anti-CIC (homemade) (Kim et al., 2015 (link)), anti-PLCγ1 (#5690, Cell Signaling), anti-p-PLCγ1 (#2821, Cell Signaling), anti-ZAP-70 (#2705, Cell Signaling), anti-p-ZAP-70 (#2701, Cell Signaling Technology), anti-JNK (#9252, Cell Signaling), anti-p-JNK (#9251, Cell Signaling), anti-ERK (#9102, Cell Signaling), anti-p-ERK (#4370, Cell Signaling), anti-p38 (#9212, Cell Signaling), anti-p-p38 (#9211, Cell Signaling), anti-β-actin (#sc-47778, Santa Cruz), and anti-α-tubulin (#sc-398103, Santa Cruz). Membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) and developed using Clarity Western ECL Substrate (Bio-Rad) or SuperSignal West Dura (Thermo Scientific). Images were acquired using an ImageQuant LAS 500 instrument (GE Healthcare).

Kim S., Park G.Y., Park J.S., Park J., Hong H, & Lee Y. (2021). Regulation of positive and negative selection and TCR signaling during thymic T cell development by capicua. eLife, 10, e71769.

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GFP-tagged protein enrichment and immunoblotting

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Cell lysates were mixed with anti-GFP monoclonal antibody coupled to agarose beads to enrich GFP-tagged proteins according to the manufacturer’s protocols (MBL). Pull-down lysates were separated by Tris-glycine PAGE, transferred to nitrocellulose membranes, and visualized as described^{20 (link)}. The following antibodies were used for biochemical assays: anti-phosphotyrosine (4G10; Millipore), anti-GFP-agarose (D153; MBL), anti-GFP (118144600; Roche), anti-pERK (9106; Cell Signaling), anti ERK (4695; Cell Signaling), anti-pSRC (2105; Cell Signaling), anti-SRC (2108; Cell Signaling), anti-pZap70 (2701; Cell Signaling), anti-actin (1616; Santa Cruz), anti-PAG (MEM-255, Origene). Prior to pERK and pSRC analysis, primary human and Jurkat T cells were stimulated for 5 min, lysed in modified 1× RIPA buffer containing protease and phosphatase inhibitors. Lysates were separated by Tris-glycine PAGE, transferred to nitrocellulose membranes, and visualized as described^{20 (link)}.

Strazza M., Azoulay-Alfaguter I., Peled M., Adam K, & Mor A. (2021). Transmembrane adaptor protein PAG is a mediator of PD-1 inhibitory signaling in human T cells. Communications Biology, 4, 672.

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