Cell Signaling Technology

Product Pathways - Tyrosine Kinase / Adaptors

Phospho-EGF Receptor Pathway Antibody Sampler Kit #9789

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb #3777 40 µl W IHC-P IF-IC F H M R Mk 175 Rabbit IgG
Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 40 µl W IP IHC-P IHC-F IF-IC F H M R Hm Mk Dm Z B (C) (X) (Dg) (Pg) 60 Rabbit IgG
Phospho-Gab1 (Tyr627) (C32H2) Rabbit mAb #3233 40 µl W H (M) (R) 110 Rabbit IgG
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 40 µl W IP IHC-P IF-IC F H M R Hm Mk Mi Dm Z B Dg Pg Sc (C) (Ce) 44, 42 Rabbit IgG
Phospho-PLCγ1 (Tyr783) Antibody #2821 40 µl W F H M R 155 Rabbit
Phospho-Shc (Tyr239/240) Antibody #2434 40 µl W H M R 50, 55, 70 Rabbit
Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb #4322 40 µl W IF-IC F H M (R) (Mk) (B) 90 Rabbit IgG
Phospho-c-Cbl (Tyr700) (D16D7) Rabbit mAb #8869 40 µl W IP H (M) 120 Rabbit
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IHC-F=Immunohistochemistry (Frozen)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  C=Chicken  Mi=Mink  Dm=D. melanogaster  X=Xenopus  Z=Zebrafish  B=Bovine  Dg=Dog  Pg=Pig  Sc=S. cerevisiae  Ce=C. elegans
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Each antibody in the Phospho-EGF Receptor Pathway Sampler Kit recognizes the phosphorylated form of its specific target. Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb may cross-react weakly with other tyrosine-phosphorylated proteins. Phospho-Gab1 (Tyr627) (C32H2) Rabbit mAb may cross-react with phosphorylated Gab2, Gab3, or activated receptor tyrosine kinases. Phospho-Shc (Tyr239/240) Antibody may cross-react with activated EGF receptor protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from Hep G2 cells, untreated or EGF-treated (100 ng/ml), and Jurkat cells, untreated or treated with anti-CD3 antibody (1 µg/ml for 10 minutes), using Phospho-Shc (Tyr239/240) Antibody #2434 (upper) or Shc Antibody #2432 (lower).

Western Blotting

Western Blotting

Western blot analysis of cell extracts from T-47D cells, untreated or stimulated with heregulin, using Phospho-Gab1 (Tyr627) (C32H2) Rabbit mAb #3233 (upper) or Gab1 Antibody #3232 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts of BxPC-3 cells, untreated or EGF-stimulated, using Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb #3777 (upper) and EGF Receptor Antibody #2232 (lower).


Western Blotting

Western Blotting

Western blot analysis of extracts from PC-3 cells, untreated or LY294002/wortmannin-treated, and NIH/3T3 cells, serum-starved or PDGF-treated, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from UT-7 cells, untreated or treated with erythropoietin (EPO; 3 units/ml for 5 min), TF-1 cells, untreated or treated with Human Granulocyte Macrophage Colony Stimulating Factor #8922 (hGM-CSF; 100 ng/ml for 10 min), and NK-92 cells, untreated or treated with Human Interleukin-2 #8907 (hIL-2; 100 ng/ml for 10 min), using Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb #4322 (upper) or total Stat5 (3H7) Rabbit mAb #9358 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from 293, NIH/3T3 and C6 cells, treated with λ phosphatase or TPA as indicated, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (upper), or p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695 (lower).


Western Blotting

Western Blotting

Western blot analysis of extracts from Jurkat cells, untreated (-) or treated with pervanadate (1 mM; +), using Phospho-c-Cbl (Tyr700) (D16D7) Rabbit mAb #8869 (upper) or c-Cbl (D4E10) Rabbit mAb #8447 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from NIH/3T3 cells, untreated or PDGF-stimulated for the indicated times, using Phospho-PLCγ1 (Tyr783) Antibody #2821 (upper) or PLCγ1 Antibody #2822 (lower).

Description

The Phospho-EGF Receptor Pathway Sampler Kit provides an economical means to evaluate the activation status of multiple members of the EGF receptor pathway, including phosphorylated EGF receptor, Stat5, c-Cbl, Shc, Gab1, PLCγ1, Akt and p44/42 MAPK. The kit includes enough primary and secondary antibodies to perform four western blot experiments.

Source / Purification

Activation state polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Tyr239/240 of human Shc and Tyr783 of human PLCγ1. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Rabbit monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Tyr700 of c-Cbl, Tyr1068 of human EGF receptor, Tyr694 of Stat5a, Tyr627 of human Gab1, Ser473 of human Akt and Thr202/Tyr204 of human p44 MAP kinase.

Background

The epidermal growth factor (EGF) receptor is a transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Ligand binding results in receptor dimerization, autophosphorylation, activation of downstream signaling, internalization, and lysosomal degradation (1,2). Phosphorylation of EGF receptor (EGFR) at Tyr845 in the kinase domain is implicated in stabilizing the activation loop, maintaining the active state enzyme, and providing a binding surface for substrate proteins (3,4). c-Src is involved in phosphorylation of EGFR at Tyr845 (5). The SH2 domain of PLCγ binds at phospho-Tyr992, resulting in activation of PLCγ-mediated downstream signaling (6). Phosphorylation of EGFR at Tyr1045 creates a major docking site for the adaptor protein c-Cbl, leading to receptor ubiquitination and degradation following EGFR activation (7,8). The GRB2 adaptor protein binds activated EGFR at phospho-Tyr1068 (9). A pair of phosphorylated EGFR residues (Tyr1148 and Tyr1173) provide a docking site for the Shc scaffold protein, with both sites involved in MAP kinase signaling activation (2). 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; mutation of either of these serines results in upregulated EGFR tyrosine autophosphorylation (10).

  1. Hackel, P.O. et al. (1999) Curr Opin Cell Biol 11, 184-9.
  2. Zwick, E. et al. (1999) Trends Pharmacol Sci 20, 408-12.
  3. Cooper, J.A. and Howell, B. (1993) Cell 73, 1051-4.
  4. Hubbard, S.R. et al. (1994) Nature 372, 746-54.
  5. Biscardi, J.S. et al. (1999) J Biol Chem 274, 8335-43.
  6. Emlet, D.R. et al. (1997) J Biol Chem 272, 4079-86.
  7. Levkowitz, G. et al. (1999) Mol Cell 4, 1029-40.
  8. Ettenberg, S.A. et al. (1999) Oncogene 18, 1855-66.
  9. Rojas, M. et al. (1996) J Biol Chem 271, 27456-61.
  10. Feinmesser, R.L. et al. (1999) J Biol Chem 274, 16168-73.

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Protocols

* Product-specific protocol.

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For Research Use Only. Not For Use In Diagnostic Procedures.

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