Western blot analysis of extracts from 293T cells expressing recombinant wild-type or mutant Syk proteins, cotransfected with CD8, using Phospho-Syk (Tyr323) Antibody (upper) or Syk Antibody #2712 (lower). (Provided by Dr. Alagarsamy L. Reddi, laboratory of Dr. Hamid Band, Harvard University, Massachusetts.)
Flow cytometric analysis of Ramos cells, untreated (blue) or treated with anti-IgM (green), using Phospho-Syk(Tyr525/526) (C87C1) Rabbit mAb, or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #8885 was used as a secondary antibody.
Two-color flow cytometric analysis of Jurkat cells, untreated (left) or anti-CD3 activated (right), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody and Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) mAb #9106. Anti-CD3 activation increases the intensity of label with both antibodies.
Flow cytometric analysis of RL-7 cells using Syk (D3Z1E) XP® Rabbit mAb (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.
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.
Western blot analysis of extracts from Ramos cells, untreated or treated with anti-human-IgM (10 µg/ml), using Phospho-Syk (Tyr323) Antibody #2715 (upper) or Syk Antibody #2712 (lower).
Confocal immunofluorescent analysis of Ramos cells, serum-starved (overnight; left) or IgM-treated (12 ug/ml, 2 minutes; right), using Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Flow cytometric analysis of Jurkat cells, untreated (blue) or CD3 treated (green), using Phospho-Zap70 (Tyr319)/Syk (Tyr352) Antibody compared to a nonspecific negative control antibody (red).
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Syk (D3Z1E) XP® Rabbit mAb.
Western blot analysis of extracts from Ramos cells, untreated or treated with anti-IgM, using Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb (upper) or Syk Antibody #2712 (lower).
Immunofluorescent analysis of Jurkat cells, CD3-treated (left) or untreated (right), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).
Immunohistochemical analysis of paraffin-embedded human lymph node using Syk (D3Z1E) XP® Rabbit mAb.
Western blot analysis of extracts from Jurkat cells, starved for 16 hours, and treated with 2 mM H2O2 or with calf intestinal alkaline phosphatase (CIP), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody (upper) or control Zap-70 Antibody #2702 (lower).
Immunohistochemical analysis of paraffin-embedded mouse spleen using Syk (D3Z1E) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
Western blot analysis of extracts from Ramos cells, untreated or treated with anti-IgM (12 µg/ ml for 2 minutes), hydrogen peroxide (10 mM for 2 minutes) or lambda phosphatase, using Phospho-Zap-70 (Tyr319)/ Syk (Tyr352) Antibody.
Immunoprecipitation of Syk protein from SR cell extracts, using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Syk(D3Z1E) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Syk (D3Z1E) XP® Rabbit mAb
Western blot analysis of extracts from various cell lines using Syk (D3Z1E) XP® Rabbit mAb.
|Phospho-Syk (Tyr323) Antibody 2715||20 µl||
|Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb 2710||20 µl||
|Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody 2701||20 µl||
||H||70 Zap-70, 72 Syk||Rabbit|
|Syk (D3Z1E) XP® Rabbit mAb 13198||20 µl||
||H M R||72||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
Phospho-Syk Sampler Kit provides an economical means to evaluate the activation status of Syk, including the phosphorylation of Tyr323, Tyr352 and Tyr525/526. The control Syk Antibody is also included. The kit contains enough primary and secondary antibodies for two Western blot experiments.
Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb detects endogenous levels of Syk protein only when phosphorylated at Tyr525/526 of human Syk or Tyr519/520 of mouse Syk. It also detects Syk protein when singly phosphorylated at Tyr526 of human Syk or Tyr520 of mouse Syk. Phospho-Syk (Tyr323) Antibody detects endogenous levels of Syk only when phosphorylated at Tyr323. Syk (D3Z1E) XP® Rabbit mAb recognizes endogenous levels of total Syk protein. All other antibodies in the kit do not cross-react with phosphorylated or nonphosphorylated forms of other related family members.
Polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Tyr323 of human Syk or Tyr319 of human Zap-70. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues around Asn463 of human Syk protein or residues surrounding Tyr525/526 of human Syk.
Syk is a protein tyrosine kinase that plays an important role in intracellular signal transduction in hematopoietic cells (1-3). Syk interacts with immunoreceptor tyrosine-based activation motifs (ITAMs) located in the cytoplasmic domains of immune receptors (4). It couples the activated immunoreceptors to downstream signaling events that mediate diverse cellular responses, including proliferation, differentiation, and phagocytosis (4). There is also evidence of a role for Syk in nonimmune cells and investigators have indicated that Syk is a potential tumor suppressor in human breast carcinomas (5). Tyr323 is a negative regulatory phosphorylation site within the SH2-kinase linker region in Syk. Phosphorylation at Tyr323 provides a direct binding site for the TKB domain of Cbl (6,7). Tyr352 of Syk is involved in the association of PLCγ1 (8). Tyr525 and Tyr526 are located in the activation loop of the Syk kinase domain; phosphorylation at Tyr525/526 of human Syk (equivalent to Tyr519/520 of mouse Syk) is essential for Syk function (9).
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