Western blot analysis of total cell lysates from DND41 and Molt4 cells using CD3ε (CD3-12) Rat mAb.
Western blot analysis of SDS extracts from untreated or anti-CD3 treated (10 µg/ml for 2 minutes) human Jurkat cells after overnight serum starvation using Phospho-LAT (Tyr191) Antibody.
Western blot analysis of extracts from Jurkat cells (starved for 16 hours) treated with calf intestinal alkaline phosphatase (CIP) or H2O2 (2 mM), using Phospho-Lck (Tyr505) Antibody (upper) or control Lck Antibody #2752 (lower).
Flow cytometric analysis of NIH/3T3 cells, untreated (blue) or treated with mPDGFbb (200ng/mL, 15 min; green) using Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Western blot analysis of extracts NIH/3T3 cells, serum-starved or treated with human Platelet-Derived Growth Factor BB hPDGF-BB #8912 (100 ng/ml, 15 min), using Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb (upper) or Src (36D10) Rabbit mAb #2109 (lower).
Western blot analysis of extracts from Jurkat cells, untreated (-) or treated with H2O2 (11 mM, 1 min; +), using Phospho-SLP-76 (Ser376) (D9D6E) Rabbit mAb (upper) or SLP-76 Antibody #4958 (lower).
Flow cytometric analysis of Ramos cells, untreated (blue) or treated with anti-IgM (green), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #8885 was used as a secondary antibody.
Western blot analysis of extracts from Ramos cells untreated or treated with 10 µg/ml IgM for 2 minutes with or without calf intestinal phosphatase (CIP), using Phospho-Zap-70 (Tyr493)/Syk (Tyr526) Antibody (upper) or Syk Antibody #12358 (lower).
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGlo® is added and emits light during enzyme catalyzed decomposition.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Immunoprecipitation of phospho-PLCγ1 (Tyr783) from A-431 cell extracts stimulated with hEGF #8916 using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb.
Western blot analysis of extracts from EL4 cells, untreated (-) or treated with H2O2 (11 mM, 1 min; +), using Phospho-SLP-76 (Ser376) (D9D6E) Rabbit mAb (upper) or SLP-76 Antibody #4958 (lower).
Confocal immunofluorescent analysis of Ramos cells, human IgM treated (left) or untreated (right), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb (green). Red = Propidium Iodide (PI)/RNase Staining Solution #4087.
Western blot analysis of extracts from Jurkat cells starved for 16 hours and treated with 2 mM H2O2 for 3 minutes, using Phospho-Zap-70 (Tyr493)/Syk (Tyr526) Antibody (upper) or control Zap-70 Antibody #2702 (lower).
Western blot analysis of extracts from NIH/3T3 cells, untreated (-) or stimulated with hPDGF-BB #8912 (5 min; +), and from A-431 cells, untreated (-) or stimulated with hEGF #8916 (5 min; +), using Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from Jurkat cells treated with hydrogen peroxide (2mM for 2 minutes) or with lambda phosphatase and extracts from Ramos cells treated with anti-human IgM (12 micrograms/ml for 2 minutes) using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb.
|CD3ε (CD3-12) Rat mAb 4443||20 µl||
||H M||21||Rat IgG1|
|Phospho-LAT (Tyr191) Antibody 3584||20 µl||
|Phospho-Lck (Tyr505) Antibody 2751||20 µl||
|Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb 14008||20 µl||
||H M||155||Rabbit IgG|
|Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb 6943||20 µl||
||H M R Mk||60||Rabbit IgG|
|Phospho-SLP-76 (Ser376) (D9D6E) Rabbit mAb 14745||20 µl||
||H M||76||Rabbit IgG|
|Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb 2717||20 µl||
||H M||70, 72||Rabbit IgG|
|Phospho-Zap-70 (Tyr493)/Syk (Tyr526) Antibody 2704||20 µl||
|Anti-rat IgG, HRP-linked Antibody 7077||100 µl||
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The T Cell Signaling Antibody Sampler Kit provides an economical means to investigate T cell receptor signaling. The kit contains primary and secondary antibodies to perform two western blot experiments per primary antibody.
Unless otherwise indicated, each antibody will recognize endogenous total levels of target protein, and modification state antibodies will only recognize target proteins phosphorylated at the indicated residue. Phospho-Lck (Tyr505) Antibody may cross-react with certain phosphorylated Src family members due to high sequence homology. Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb may cross-react with other Src family members (Lyn, Fyn, Lck, Yes and Hck) when phosphorylated at equivalent sites, and may cross react with overexpressed phosphorylated RTKs. Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb cross-reacts with endogenous levels of Syk when phosphorylated at Tyr352. Phospho-Zap-70 (Tyr493)/Syk (Tyr526) Antibody cross-reacts with endogenous levels of Syk when phosphorylated at Tyr526.
Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to a region surrounding Pro184 of human CD3ε, and with a synthetic phosphopeptide corresponding to residues surrounding Tyr783 of human PLCγ1 protein, Tyr416 of human Src protein, Ser376 of human SLP-76 protein, and Tyr319 of human Zap-70 protein. Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Tyr191 of human LAT protein, Tyr505 of human Lck protein, and Tyr493 of human Zap-70 protein. Antibodies are purified by protein A and peptide affinity chromatography.
When T cells encounter antigens via the T cell receptor (TCR), information about the quantity and quality of antigens is relayed to the intracellular signal transduction machinery (1). This activation process depends mainly on CD3 (Cluster of Differentiation 3), a multiunit protein complex that directly associates with the TCR α and ß chains. CD3 is composed of four polypeptides: ζ, γ, ε and δ. Each of these polypeptides contains at least one immunoreceptor tyrosine-based activation motif (ITAM) (2). The Src family kinases Lck and Fyn are recruited to the TCR complex upon stimulation and activate the downstream tyrosine kinases to initiate signaling. Phosphorylation of Lck at Tyr394 leads to an increase in Lck activity while phosphorylation of Tyr505 in the Lck carboxy-terminal tail down-regulates Lck catalytic activity (3). Zap-70 and Syk are rapidly phosphorylated on several tyrosine residues through autophosphorylation and transphosphorylation by Src family tyrosine kinases. Activation loop phosphorylation of Zap-70 at Tyr493 and Syk at Tyr526 leads to complete activation of both kinases (4). Subsequent phosphorylation of other tyrosine residues within the kinase interdomain B region, including Zap-70 at Tyr315 and Zap-70 at Tyr 319, create docking sites for downstream signaling molecules. Zap-70 and Syk phosphorylate the transmembrane adaptor protein LAT at multiple, conserved tyrosine residues within SH2 binding motifs, exposing these motifs as docking sites for downstream signaling targets (5,6). The phosphorylation of LAT at Tyr171 and Tyr191 enables the binding of Grb2, Gads/SLP-76, PLCγ1, and PI3 kinase. The adapter protein SLP-76 is phosphorylated at Tyr113 and Tyr128, allowing for binding of the Grb2-like adapter Gads. Phosphorylation of SLP-76 at Ser376 by hematopoietic progenitor kinase 1 (HPK1) induces interaction with 14-3-3ε and down-regulates TCR signaling (7,8). Phosphoinositide-specific phospholipase PLCγ1 enzyme activity is also stimulated by Zap-70 and Syk phosphorylation on Tyr783, Tyr711, and Tyr1253, resulting in robust PI-4,5-P2 hydrolysis (9).
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