Anti zap70

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-ZAP70 is a laboratory reagent used to detect the presence and quantity of the ZAP70 protein in cellular samples. ZAP70 is a key signaling molecule involved in T-cell activation and function.

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Lab products found in correlation

Anti slp 76, by Cell Signaling Technology (3 mentions) Anti p erk, by Cell Signaling Technology (2 mentions) Anti pzap70, by Cell Signaling Technology (2 mentions) Anti plcγ1, by Cell Signaling Technology (2 mentions) Anti lat, by Cell Signaling Technology (2 mentions) Secondary antibodies for immunoblotting, by Merck Group (1 mentions) Anti cd3ε m20, by Santa Cruz Biotechnology (1 mentions) Anti cd3ε okt3, by Thermo Fisher Scientific (1 mentions) Anti gapdh, by Merck Group (1 mentions) Anti lck, by Cell Signaling Technology (1 mentions) Anti mouse cd4, by Thermo Fisher Scientific (1 mentions) Anti erk, by BD (1 mentions) Anti human cd8, by Thermo Fisher Scientific (1 mentions) Anti human cd3, by BD (1 mentions) Anti mouse cd4, by BD (1 mentions) Anti mouse cd4, by BioLegend (1 mentions) Anti human cd8, by BD (1 mentions) Anti rna polymerase 2, by Abcam (1 mentions) Anti mouse cd8, by BD (1 mentions) Anti mouse cd25, by BD (1 mentions)

Spelling variants of this product

ZAP70 (20 citations) Anti-phospho-Zap70 (Tyr319) (3 citations) Anti-ZAP70-pTyr319 (2 citations) Anti-pY493 ZAP70 (2 citations)

10 protocols using anti zap70

T Cell Receptor Signaling Antibodies

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The following antibodies were used: anti-Nck1, anti-ZAP70, anti-Lck, anti-phospho-Lck (Src416) and anti-phospho-ZAP70 (Y319, Cell Signaling Technology), anti-phospho-ζ (Y142, Sigma-Aldrich), anti-idiotypic TCR (C305, Millipore), anti-CD3ε (M20, Santa Cruz Biotechnology), anti-CD3ε (OKT3, eBioscience), anti-ζ antiserum 448 [4 (link)], anti-phospho-CD3ε (Y188) [30 (link)], anti-GST (Bethly), anti-GAPDH (Sigma) and secondary antibodies for immunoblotting (Perbio). Alexa Fluor 647-labeled anti-CD3ε (UCHT1, BioLegend) was used for flow cytometry. Recombinant His-tagged Nck was from Sigma and recombinant active Lck (aa61-aa509) was a generous gift from B.F. Lillemeier, Salk Institute for Biological Studies, San Diego.

Hartl F.A., Ngoenkam J., Beck-Garcia E., Cerqueira L., Wipa P., Paensuwan P., Suriyaphol P., Mishra P., Schraven B., Günther S., Pongcharoen S., Schamel W.W, & Minguet S. (2021). Cooperative Interaction of Nck and Lck Orchestrates Optimal TCR Signaling. Cells, 10(4), 834.

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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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Phosphorylation of Zap-70 in Activated Mouse T Cells

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Isolated mouse T cells were obtained as described above. One million cells were resuspended in 30 μl of 10% FBS medium or 10% delipidated FBS medium with CD3/CD28 activation beads in a bead-to-cell ratio of 1∶1. After 2 min of incubation, cells were mixed by pipetting up and down 5 times in RIPA buffer (Sigma-Aldrich) containing complete, Mini protease inhibitor cocktail tablet (Roche Diagnostics) Proteins were extracted by freeze and thaw three times and then centrifuged at 13793 g for 20 min at 4°C. Protein concentrations were determined by Pierce BCA Protein Assay Kit (Thermo Scientific). Ten μg of protein were loaded on a 12% SDS-Page gel and then transferred to nitrocellulose membrane. The membrane was blocked with 5% BSA and then probed with anti phospho-Zap-70, anti Zap-70 or anti α-tubulin (Cell Signaling) as primary antibody and ECL HRP labelled anti rabbit IgG (GE Healthcare) as secondary antibody. Amersham ECL Prime detection reagent (GE Healthcare) was used to visualize protein bands.

Chyu K.Y., Lio W.M., Dimayuga P.C., Zhou J., Zhao X., Yano J., Trinidad P., Honjo T., Cercek B, & Shah P.K. (2014). Cholesterol Lowering Modulates T Cell Function In Vivo and In Vitro. PLoS ONE, 9(3), e92095.

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Jurkat T-cell Line Variants and Receptor Expression

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The Jurkat E6.1 T-cell line and its Zap70- (P116) and Lat-(JCaM2.5) deficient variants were kindly provided by A. Weiss (UCSF, San Francisco, USA). A chimeric cDNA construct coding for the extracellular and transmembrane regions of human CD25 linked to the cytoplasmic region of mouse CD3ξ and termed CD25ξ has been described^{50 (link)}. A full-length cDNA corresponding to the largest isoform of human CD6 (CD6-201 Ensembl ENST00000344028) was isolated from Jurkat cells by reverse transcription and cloned into the pEF6 vector (Life Technology). Stable transfectants expressing CD25ξ chimeric constructs and human CD6 molecules were obtained by electroporation of Jurkat cells and sorted based on their level of CD6 or CD25ξ expression. Stimulating antibodies against CD25 (7G7) were from Millipore. Anti-ZAP70 (2705; Cell Signaling Technology), anti-SLP76 (4958; Cell Signaling Technology), anti-LAT (06-807; Millipore), anti-GADS (06-983; Millipore), anti-CD6 (H300; Santa Cruz Biotechnology), and antibodies to phosphorylated tyrosines (4G10; Millipore) were used for immunoblot analysis.

Roncagalli R., Hauri S., Fiore F., Liang Y., Chen Z., Sansoni A., Kanduri K., Joly R., Malzac A., Lähdesmäki H., Lahesmaa R., Yamasaki S., Saito T., Malissen M., Aebersold R., Gstaiger M, & Malissen B. (2014). Quantitative proteomic analysis of signalosome dynamics in primary T cells identifies the CD6 surface receptor as a Lat-independent TCR signaling hub. Nature immunology, 15(4), 384-392.

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Immunoblot Analysis of Cell Signaling

Cited 1 time

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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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Stimulation of Mouse CD4+ T Cells

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Mouse CD4⁺ T cells from WT C57BL/6 or TRAIL-R-KO mice were enriched (STEMCELL Technologies) and stimulated at the indicated time point at 37°C with medium, an anti-CD3 Ab (1 µg/ml), an anti-CD28 Ab (1 µg/ml), and the TRAIL (10 µg/ml), or a combination of anti-CD3/anti-CD28 Abs and the TRAIL. Whole-cell protein was subsequently extracted using the PhosphoSafe Extraction Reagent (Merck Millipore, Darmstadt, Germany). Protein lysates were transferred onto a polyvinylidene difluoride membrane after sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and labeled using monoclonal primary Abs against anti-phospho-ZAP70 (Tyr319) (Cell Signaling, Beverly, MA, USA), anti-ZAP70 (Cell Signaling), anti-phospho-LAT (Tyr191) (Abcam, Cambridge, UK), anti-LAT (Thermo Scientific), anti-phospho-PLCγ1 (Tyr783) (Cell Signaling), anti-PLCγ1 (Abcam), and anti-β-actin (Abcam) Abs. The secondary Ab was labeled with HRP, and electrochemiluminescence was used to visualize the bands.

Chyuan I.T., Tsai H.F., Wu C.S., Sung C.C, & Hsu P.N. (2018). TRAIL-Mediated Suppression of T Cell Receptor Signaling Inhibits T Cell Activation and Inflammation in Experimental Autoimmune Encephalomyelitis. Frontiers in Immunology, 9, 15.

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Antibody Panel for Signaling Pathways

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Anti-SHP-1 was purchased from Abcam. Anti-AMPK, anti-phosphorylated-AMPK, anti-ZAP70, anti-phosphorylated-ZAP70, anti-mTOR, anti-phosphorylated-mTOR, anti-p70 S6 kinase, and anti-phosphorylated-p70 S6 kinase antibodies were acquired from Cell Signaling Technology.

Kuwabara T., Ishikawa F., Ikeda M., Ide T., Kohwi-Shigematsu T., Tanaka Y, & Kondo M. (2021). SATB1-dependent mitochondrial ROS production controls TCR signaling in CD4 T cells. Life Science Alliance, 4(11), e202101093.

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Western Blot Analysis of T Cell Signaling Proteins

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Equal masses of protein lysate were loaded into 4–12% Bis-Tris NuPAGE Gels (ThermoFisher). After protein transfer onto nitrocellulose membranes (ThermoFisher), membranes were blocked with Western Blocking Reagent (Sigma) diluted 1:10 in 1× Tris-buffered saline (TBS). Membranes were stained with primary and secondary antibodies diluted 1:5,000–1:10,000 in SuperBlock TBS (ThermoFisher) supplemented with 0.1% Tween. The following antibodies were used: anti-CD247 (8D3, BD Biosciences), anti-CD247 pTyr¹⁴² (K25–407.69, BD Biosciences), anti-LAT (polyclonal, Cell Signaling), anti-LAT pTyr²²⁰ (polyclonal, Cell Signaling), anti-LCK (D88, Cell Signaling), anti-NCK1 (15B9, Cell Signaling), anti-PLC-γ1 (D9H10, Cell Signaling), anti-PLC-γ1 pTyr⁷⁸³ (D6M9S, Cell Signaling), anti-PLC-γ1 pSer¹²⁴⁸ (D25A9, Cell Signaling), anti-SLP-76 (polyclonal, Cell Signaling), anti-SLP-76 pSer³⁷⁶ (D9D6E, Cell Signaling), anti-ZAP-70 (D1C10E, Cell Signaling), anti-ZAP-70 pTyr³¹⁹ (65E4, Cell Signaling), anti-ZAP-70 pTyr⁴⁹³ (polyclonal, Cell Signaling), anti-mouse horseradish peroxidase (HRP) (polyclonal, Cell Signaling), and anti-rabbit HRP (polyclonal, Cell Signaling). Band intensities were quantified using ImageJ [National Institutes of Health (NIH)]; normalized to total protein or loading control, and then renormalized to a control sample.

Salter A.I., Rajan A., Kennedy J.J., Ivey R.G., Shelby S.A., Leung I., Templeton M.L., Muhunthan V., Voillet V., Sommermeyer D., Whiteaker J.R., Gottardo R., Veatch S.L., Paulovich A.G, & Riddell S.R. (2021). Comparative analysis of TCR and CAR signaling informs CAR designs with superior antigen sensitivity and in vivo function. Science signaling, 14(697), eabe2606.

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Immunofluorescence Staining of Cell Markers

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Cells were fixed in 4% paraformaldehyde in PBS for 15 min, subjected to three 5 min washes with PBS, and incubated in blocking buffer (5% normal goat serum, 0.3% Triton X-100 in PBS) for 1 hr at room temperature. Samples were then incubated in antibody dilution buffer (2% bovine serum albumin, 0.3% Triton X-100 in PBS) containing primary antibodies overnight at 4°C. The following antibodies were used: anti-EEA1 (Cell Signaling Technology 48453); anti-ZAP70 (Cell Signaling Technology 3165). The next day, samples were subjected to three 5 min washes with PBS, incubated in antibody dilution buffer containing Alexa Fluor-conjugated secondary antibodies for 1 hr at room temperature, and subjected to another three 5 min washes with PBS. Samples were then imaged by confocal microscopy on a Nikon Eclipse Ti microscope as described above.

Farahani P.E., Yang X., Mesev E.V., Fomby K.A., Brumbaugh-Reed E.H., Bashor C.J., Nelson C.M, & Toettcher J.E. (2023). pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells. eLife, 12, e82863.

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Immunoblot analysis of T-cell signaling

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Affinity-purified samples and whole-cell lysates were loaded on 8–10% SDS-PAGE gel and subsequently analyzed by immunoblot with specific antibodies. The following antibodies were used for immunoblot analysis: anti–ZAP-70 (2705; Cell Signaling Technology), anti-LAT (9166; Cell Signaling Technology), anti–SLP-76 (4958; Cell Signaling Technology), anti-VAV1 (2502; Cell Signaling Technology), and anti-phosphotyrosine (4G10; Millipore).

Nicolas P., Ollier J., Mori D., Voisinne G., Celis-Gutierrez J., Gregoire C., Perroteau J., Vivien R., Camus M., Burlet-Schiltz O., Gonzalez de Peredo A., Clémenceau B., Roncagalli R., Vié H, & Malissen B. (2022). Systems-level conservation of the proximal TCR signaling network of mice and humans. The Journal of Experimental Medicine, 219(2), e20211295.

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