Immunohistochemical analysis of paraffin-embedded SK-BR-3 cell pellets, either untreated (upper) or EGF-treated (lower), using Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb #3777, Phospho-HER2/ErbB2 (Tyr1221/1222) (6B12) Rabbit mAb #2243, Phopho-HER3/ErbB3 (Tyr1289) (D1B5) Rabbit mAb #2842 or EGF Receptor (D38B1) XP® Rabbit mAb #4267. Cell pellets are provided in the SignalSlide® ErbB Family IHC Controls.
Schematic of placement of cell pellets on SignalSlide® Phospho-ErbB Family IHC Controls #8117.
|8111S||1 Kit (4 x 40 µl)||N/A|
|Kit Includes||Quantity||Antigen Retrieval / Diluent||Isotype|
|Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb #3777||40 µl||EDTA / SignalStain® Antibody Diluent #8112||Rabbit IgG|
|Phospho-HER2/ErbB2 (Tyr1221/1222) (6B12) Rabbit mAb #2243||40 µl||EDTA / SignalStain® Antibody Diluent #8112||Rabbit IgG|
|Phospho-HER3/ErbB3 (Tyr1289) (D1B5) Rabbit mAb #2842||40 µl||EDTA / SignalStain® Antibody Diluent #8112||Rabbit IgG|
|EGF Receptor (D38B1) XP® Rabbit mAb #4267||40 µl||EDTA / SignalStain® Antibody Diluent #8112||Rabbit IgG|
|*SignalStain® Antibody Diluent #8112||25 ml|
|†SignalSlide® Phospho-ErbB Family IHC Controls #8117||1 Pack|
*SignalStain® Antibody Diluent is supplied as a working solution and should be stored at 4ºC (packaged separately).
†Control slides should be stored at 4ºC (packaged separately).
The SignalStain® Phospho-ErbB Family IHC Sampler Kit from Cell Signaling Technology allows the researcher to examine paraffin-embedded tissues or cells with antibodies that will detect active ErbB 1, 2 and 3 as well as total epidermal growth factor receptor (EGFR). Each antibody is validated for use in immunohistochemical assays using multiple approaches. Also included in the kit are control slides that can be used to verify the performance of each antibody and a primary antibody diluent. See the table above for the recommended antibody diluent for each antibody provided in the kit.
Each antibody in the SignalStain® Phospho-ErbB Family IHC Sampler Kit detects endogenous levels of its target protein. Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb detects endogenous EGF receptor only when phosphorylated at Tyr1068. This antibody may cross-react weakly with other tyrosine-phosphorylated proteins. Phospho-HER2/ErbB2 (Tyr1221/1222) (6B12) Rabbit mAb detects endogenous levels of ErbB2 only when phosphorylated at Tyr1221/1222. The antibody does not cross react with other activated ErbB family members or other tyrosine-phosphorylated proteins. Phopho-HER3/ErbB3 (Tyr1289) (D1B5) Rabbit mAb detects endogenous levels of HER3/ErbB3 proteins only when phosphorylated at tyrosine 1289. The antibody does not cross-react with other phosphorylated receptor tyrosine kinases at significant level. EGF Receptor (D38B1) Rabbit mAb detects endogenous levels of total EGF receptor protein.
Monoclonal antibody is produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Tyr1068 of human EGFR, Tyr1221/1222 of human ErbB2, or to Tyr1289 of human HER3/ErbB3. Monoclonal antibody to EGFR is produced by immunizing animals with a synthetic GST-fusion protein corresponding to residues containing the cytoplasmic domain of human EGF receptor.
The epidermal growth factor (EGF) receptor is a 170 kDa transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Ligand binding results in receptor dimerization, autophosphorylation, activation of downstream signaling and lysosomal degradation (1,2). EGFR is phosphorylated on multiple tyrosine residues, each of which leads to activation of a specific downstream pathway. Major residues involved in EGFR signaling include: Tyr845, Tyr992, Tyr1045, Tyr1068, Tyr1148 and Tyr1173 (2-9). Phosphorylation of EGFR at specific serine and threonine residues attenuates EGFR kinase activity. EGFR carboxy-terminal residues Ser1046 and Ser1047 are phosphorylated by CaM kinase II; a mutation to either of these serines results in upregulated EGFR tyrosine autophosphorylation (10).
The ErbB2 (HER2) proto-oncogene encodes a 185 kDa transmembrane, receptor-like glycoprotein with intrinsic tyrosine kinase activity (11). While ErbB2 lacks an identified ligand, ErbB2 kinase activity can be activated in the absence of a ligand when overexpressed and through heteromeric associations with other ErbB family members (12). Amplification of the ErbB2 gene and overexpression of its product are detected in almost 40% of human breast cancers, as such it is a key therapeutic target (13). ErbB2 has several key residues that are phosphorylated upon its activation including Tyr877, Tyr1221/1222 and Tyr1248 (11,14).
HER3/ErbB3 is a member of the ErbB receptor protein tyrosine kinase family, but lacks tyrosine kinase activity. Tyrosine phosphorylation of ErbB3 depends on its association with other ErbB tyrosine kinases. Ligand binding promotes formation of a heterodimer containing ErbB3 and another ErbB protein and subsequent tyrosine phosphorylation of ErbB3 by the activated ErbB kinase (15,16). At least nine putative carboxy-terminal tail tyrosine phosphorylation sites are found in ErbB3, including Tyr1222 and Tyr1289 (17). ErbB3 may function as an oncogenic unit together with other ErbB members in tumor development; ErbB2 requires ErbB3 to drive breast tumor cell proliferation (18). A novel anti-tumor strategy involves inhibiting the interaction between ErbB3 and ErbB tyrosine kinases.
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