Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

Flow Cytometry analysis of Jurkat (human acute T cell leukemia) treated (Red)/untreated (Green) with 1mM pervanadate for 4 hours with purified ab68236 at 1/250 dilution. The secondary antibody was Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution. A Rabbit monoclonal IgG (Black) was used as the isotype control and cells without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

Immunocytochemistry/Immunofluorescence analysis of Jurkat cells (untreated, Per treated and Per+LP treated) labelling CD3 zeta (phospho Y83) with ab68236 (left) and CD3 zeta with ab40804 (right) both at a dilution of 1/200. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

The image shows increased cytoplamic staining after Pervanadate (1 mM, 30 min) treatment on Jurkat cells. The LP treatment decreased the cytoplasmic staining caused by Pervanadate.

ab40804 was used as a Pan control for ab68236. The results showed cytoplamic staining on untreated, pervanadate (1 mM, 30 min) treated and Per+LP treated Jurkat cells.

• IP

Lab

Immunoprecipitation - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

CD3 zeta was immunoprecipitated from 0.35 mg Jurkat (Human T cell leukemia T lymphocyte) treated with pervandate (50mM 5min) whole cell lysate 10 μg with ab68236 at 1/30 dilution (2μg) . VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) treated with pervandate (50mM 5min) whole cell lysate 10 μg

Lane 2 : ab68236 IP in Jurkat treated with pervandate (50mM 5min) whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab68236 in Jurkat treated with pervandate (50mM 5min) whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (ab68236)

Predicted band size: 19 kDa

Observed band size: 18-22 kDa

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• WB

Lab

Western blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

Blocking buffer 5% NFDM/TBST

Diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (ab68236) at 1/2000 dilution

Lane 1:

Untreated Jurkat cells whole cell lysates at 10 µg

Lane 2:

Jurkat cells were treated with 50mM Pervanadate for 5 minutes whole cell lysates at 10 µg

Lane 3:

Jurkat cells were treated with 50mM Pervanadate for 5 minutes whole cell lysates, then the membrane was incubated with Alkaline phosphatase at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 19 kDa

Observed band size: 18 kDa

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Exposure time: 3min

• WB

Unknown

Western blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

All lanes:

Western blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (ab68236) at 1/10000 dilution

Lane 1:

Jurkat cell lysate, untreated. at 10 µg

Lane 2:

Jurkat cell lysate, treated with pervanadate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 19 kDa

Observed band size: 18-22 kDa

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• Dot

Unknown

Dot Blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

Dot blot analysis of CD3 zeta (pY83) phospho peptide (lane 1) and CD3 zeta non-phospho peptide (lane 2) labelling CD3 zeta (phospho Y83) with ab68236 at a dilution of 1/1000. A peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/2500).

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

• WB

CiteAb

Western blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

Western Blotting using Recombinant Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y], ab68236. Publication image from Jorge, I. et al., 2016, Nat Commun, 27091106. Legend direct from paper.

Aurora A inhibition does not affect actin cytoskeleton dynamics.(a) Immunoblot of a pull-down assay of GST-Nck fusion protein from cell lysates of control (DMSO; vehicle) or Aurora A inhibitor (MLN8237)-pretreated human T lymphoblasts. Activation was performed with soluble anti-CD3ɛ antibodies for indicated times. CD3ζ and GST are shown. CD3ζ content in whole-cell lysates is indicated in the bottom row. (b) Immunoblotting of Rac1 pull-down assay of GST and GST-PAK-CD from cell lysates of DMSO- or MLN8237-pretreated Jurkat T cells activated with SEE-pulsed Raji B cells (APCs) for the indicated times. Loading control for Rac1 in whole-cell lysates is shown. (c) Images from TIRFm time-lapse analysis of mCherry–β-actin-expressing Jurkat T cells spreading over anti-CD3/CD28-coated glass-bottom chambers. Cells were pretreated with DMSO or MLN8237. Images were taken every 100 ms for 5 min at 90 nm penetrance. A corresponding bright-field image is shown. Scale bar, 10 µm. (d) Quantification of the area occupied by the whole cell (lamella), the actin-rich area (peripheral SMAC (pSMAC)), the central area (cSMAC) and the distribution of mean fluorescence intensity per area (ratios cSMAC : cell; pSMAC : cell and cSMAC/pSMAC) from cells in c (n=48 and n=36, three independent experiments). Cells were fixed after spreading (4 min) and fluorescence images were taken. Data represent means±s.d.; t-test. n.s., nonsignificant. (e) Maximum Z projections of confocal stacks from DMSO- or MLN8237-pretreated Jurkat T cells conjugated with SEE-APCs. Cells were incubated for 30 min, fixed and stained forα-tubulin (green) or actin (magenta). The right-hand image shows CMAC cell tracker labelling of APCs (cyan) and bright field. Scale bar, 10 µm. (f) Quantification of actin accumulation at the IS contact area in conjugates as in e from three independent experiments (n=100). Data represent means±s.d.; t-test. (g) Image sequence for IS formation between mCherry–β-actin-expressing T cells and SEB-APCs (DMSO- or MLN8237-treated). XYZ stacks were acquired every 25 s (maximal projections of XYZ stacks and 3D reconstructions with Imaris Software are shown from representative conjugates). (h) Ratio of T cells forming lamella on contact with an APC from g. Data represent median±interquartile range. Mann–Whitney test (DMSO : 28 cells (n=4); MLN8237 : 25 cells (n=3)).

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• WB

CiteAb

Western blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

Western Blotting using Recombinant Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y], ab68236. Publication image from Jorge, I. et al., 2016, Nat Commun, 27091106. Legend direct from paper.

Aurora controls localization and phosphorylation of the tyrosine kinase Lck.(a) Maximum Z projection of XYZ stack of human Jurkat T cells pretreated with vehicle (DMSO) or Aurora A inhibitor (MLN8237) and conjugated with SEE-preloaded Raji B cells (APCs; 30 min). Cells were fixed and stained forα-tubulin–fluorescein isothiocyanate (FITC) (green), PKCθ (magenta) and Lck (red). Bright-field images are included. Scale bar, 10 µm. (b) Quantification of Lck accumulation at the IS in conjugates as in a from three independent experiments (DMSO, n=96. MLN8237, n=94). Data represent means±s.d. Means were compared with a t-test; ****P<0.0001. (c) Immunoblotting of Lck phosphorylation at Y394 in primary human CD4+ T cells. Cells were pretreated with DMSO or MLN8237 and conjugated for the indicated times with anti-CD3/CD28-coated beads. Total Lck and actin are included as loading controls. Arrows point Lck band. (d) Quantification of data from four independent experiments as in c. Error bars represent s.d. Medians were compared with a Friedman test (*P<0.05). (e) Immunoblots of CD3ζ phosphorylation in lysates of J.CAM1 T cells transfected with Lck-GFP, CD4-Lck or CD4-δCyt-GFP, pretreated with DMSO or MLN8237 and conjugated for 5 min with SEE-pulsed APCs. (f) T-cell lymphoblasts pretreated with DMSO or MLN8237 were activated or not with SEE-pulsed APCs (2 min) and subjected to IP using an anti-Lck antibody. The immunoprecipitates were subjected to MS analysis. Upper panel, MS/MS extracted ion chromatograms of the Y394-phosphorylated and non-modified forms of Lck peptide LIEDNEYTAR. Lower panel, phosphorylated : non-modified peak ratios. (g) Recombinant Lck was incubated with Aurora A WT (in the absence or presence of MLN8237) or Aurora A KD immunoprecipitated from nocodazole-treated (16 h), transfected HEK293 cells. Lck and Aurora were incubated for 30 min in the presence of ATP and the mixture analysed by MS. Upper panel, MS/MS extracted ion chromatograms of the Y394-phosphorylated and non-modified forms of Lck peptide LIEDNEYTAR. Lower panel, phosphorylated : non-modified peak ratios. See Supplementary Table 1 for representative MS/MS spectra of the phosphorylated and non-phosphoryated forms of the peptide at the peaks.

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• WB

CiteAb

Western blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

Western Blotting using Recombinant Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y], ab68236. Publication image from Jorge, I. et al., 2016, Nat Commun, 27091106. Legend direct from paper.

Aurora A inhibition impairs TCR signalling pathways.(a) Immunoblottings showing phosphorylation of the indicated molecules in lysates of J77 Jurkat T cells pretreated with vehicle (DMSO) or Aurora A inhibitor (MLN8237) and conjugated for the indicated times with SEE-pulsed Raji B cells. (b) Quantification of blots as in a from four to six independent experiments. Error bars represent interquartile range. Medians were compared with a Friedman test (*P<0.05). n.s., nonsignificant. (c) Immunoblottings showing phosphorylation of the indicated molecules in lysates of DMSO- or MLN8237-pretreated primary human CD4+ T cells conjugated for the indicated times with anti-CD3/CD28-coated beads. (d) Quantification of blottings as in c from four to six independent experiments. Error bars represent interquartile range. Medians were compared with a Friedman test (*P<0.05). n.s., nonsignificant.

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• WB

CiteAb

Western blot - Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y] (AB68236)

Western Blotting using Recombinant Anti-CD3 zeta (phospho Y83) antibody [EP776(2)Y], ab68236. Publication image from Jorge, I. et al., 2016, Nat Commun, 27091106. Legend direct from paper.

Aurora A inhibition impairs TCR signalling and gene expression.(a) Immunoblottings showing phosphorylation of the molecules indicated in lysates of CH7C17 Jurkat T cells pretreated with DMSO or MLN8237 and conjugated for the indicated times with HA-peptide-pulsed Hom2 B cells. (b) Quantification of blots as in a–c from four to six independent experiments. Error bars represent interquartile range. Medians were compared with a Friedman test (*P<0.05). n.s., nonsignificant. (c) IL2, CD69 and CD25 mRNA levels in primary human CD4+ T cells pretreated with DMSO, MLN8237 (10 µM) or the Aurora B inhibitor AZD1152 (100 nM) and activated by settling on anti-CD3/CD28-coated plates for 4 h. mRNA levels were normalized to the housekeeping gene GAPDH and the levels of the target mRNA in non-stimulated cell levels. Error bars represent interquartile rage. Medians were compared with a Mann–Whitney test. **P<0.01.

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