Immunoprecipitation of Phospho-Btk (Tyr223) from Ramos cells, serum-starved followed by treatment with anti-human IgM (12 μg/ml, 10 min) using Phospho-Btk (Tyr223) (D1D2Z) Rabbit mAb. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotyope Control #3900, lane 3 is Phospho-Btk (Tyr223) (D1D2Z) Rabbit mAb (1:50), and lane 4 is Phospho-Btk (Tyr223) (D1D2Z) Rabbit mAb (1:100). Western blot analysis was performed using Phospho-Btk (Tyr223) (D1D2Z) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
Human whole blood was fixed, lysed, and permeabilized as per the Cell Signaling Technology Flow Alternate Protocol and stained using Btk (D3H5) Rabbit mAb. Samples were co-stained using CD3-PE and CD19-APC to distinguish T and B cell subpopulations, respectively. B (red) and T (blue) cell population gates were applied to a histogram depicting the mean fluorescence intensity of Btk. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using Btk (D3H5) Rabbit mAb performed on the Leica® Bond™ Rx.
Western blot analysis of extracts from Daudi cells, serum-starved overnight, then vehicle-treated (lane 1), treated with anti-human IgM (12 μg/ml, 10 min; lane 2), or pre-treated with Ibrutinib #16483 (1 μM, 60 min) prior to anti-IgM treatment (lane 3), using Phospho-Btk (Tyr23) (D1D2Z) Rabbit mAb (upper) or Btk (D6H5) Rabbit mAb #8547 (lower).
Flow cytometric analysis of Daudi cells (green) and Jurkat cells (blue) using Btk (D3H5) Rabbit mAb (solid lines) or a 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.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Btk (D3H5) Rabbit mAb. Note staining of inflammatory cells.
Immunohistochemical analysis of paraffin-embedded human B-cell lymphoma using Btk (D3H5) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded mouse colon using Btk (D3H5) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Btk (D3H5) Rabbit mAb. Note staining of inflammatory cells.
Immunohistochemical analysis of paraffin-embedded cell pellets, Ramos(left) or Jurkat (right), using Btk (D3H5) Rabbit mAb.
Western blot analysis of extracts from various cell lines using Btk (D3H5) Rabbit mAb.
PhosphoPlus® Duets from Cell Signaling Technology (CST) provide a means to assess protein activation status. Each Duet contains an activation-state and total protein antibody to your target of interest. These antibodies have been selected from CST's product offering based upon superior performance in specified applications.
Bruton's tyrosine kinase (Btk) is a member of the Btk/Tec family of cytoplasmic tyrosine kinases. Like other Btk family members, it contains a pleckstrin homology (PH) domain and Src homology SH3 and SH2 domains. Btk plays an important role in B cell development (1,2). Activation of B cells by various ligands is accompanied by Btk membrane translocation mediated by its PH domain binding to phosphatidylinositol-3,4,5-trisphosphate (3-5). The membrane-localized Btk is active and associated with transient phosphorylation of two tyrosine residues, Tyr551 and Tyr223. Tyr551 in the activation loop is transphosphorylated by the Src family tyrosine kinases, leading to autophosphorylation at Tyr223 within the SH3 domain, which is necessary for full activation (6,7). The activation of Btk is negatively regulated by PKCβ through phosphorylation of Btk at Ser180, which results in reduced membrane recruitment, transphosphorylation, and subsequent activation (8). The PKC inhibitory signal is likely to be a key determinant of the B cell receptor signaling threshold to maintain optimal Btk activity (8).
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