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9789
Phospho-EGF Receptor Pathway Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Phospho-EGF Receptor Pathway Antibody Sampler Kit #9789

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Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 1

Flow cytometric analysis of A549 cells, untreated (blue) or EGF-treated (green), using Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb compared to concentration matched XP® Rabbit (DA1E) mAb IgG Isotype Control #3900 (red).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 2

Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, Wortmannin #9951, and U0126 #9903 (50 μM, 1 μM, and 10 μM, 2 hr; blue) using Phospho-Akt (Ser473) (D9E) XP® 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.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 3

Western blot analysis of cell extracts from T47D cells, untreated or stimulated with heregulin, using Phospho-Gab1 (Tyr627) (C32H2) Rabbit mAb (upper) or Gab1 Antibody #3232 (lower).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 4

Flow cytometric analysis of Jurkat cells, treated with U0126 (10 µM, 2 hrs; green) or treated with TPA #4174 (200 nM, 30 min; blue) using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® 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.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 5

Western blot analysis of extracts from HepG2 cells, untreated or EGF-treated (100 ng/ml) 18 hours of serum-starvation, and Jurkat cells, untreated or treated with anti-CD3 antibody (1 µg/ml for 10 minutes), using Phospho-Shc (Tyr239/240) Antibody (upper) or Shc antibody #2432 (lower).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 6

Flow cytometric analysis of TF-1 cells, untreated (blue) or GM-CSF treated (green), using Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 7

Immunoprecipitation (IP) of phosphorylated c-Cbl from Jurkat cell extracts, untreated (-) or treated with pervanadate (1 mM; +), using Phospho-c-Cbl (Tyr700) (D16D7) Rabbit mAb (lanes 4 and 6). Western blot detection was performed using the same antibody. Lanes 1 and 2 are 10% input. Lanes 3 and 5 are IPs performed with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 8

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.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 9

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.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 10

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or EGF-treated (right), using Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 11

Confocal immunofluorescent analysis of C2C12 cells, LY294002-treated (left) or insulin-treated (right), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin #8953 (red). Blue pseudocolor = DRAQ5®#4084 (fluorescent DNA dye).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 12

Confocal immunofluorescent analysis of Drosophila egg chambers, untreated (top) or λ phosphatase-treated (bottom), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (green) and S6 Ribosomal Protein (54D2) Mouse mAb #2317 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 13

Confocal immunofluorescent analysis of A-431 cells, EGF-treated (left) or untreated (right), using Phospho-Stat5 (Tyr694) XP®(D47E7) Rabbit mAb (green) and Pan-Keratin (C11) Mouse mAb #4545 (red).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 14

Western blot analysis of extracts from Jurkat cells, untreated (-) or treated with pervanadate (1 mM; +), using Phospho-c-Cbl (Tyr700) (D16D7) Rabbit mAb (left). The phospho-specificity of the antibody was verified by preincubating the antibody with c-Cbl (Tyr700) phosphopeptide (center) or with c-Cbl (Tyr700) nonphosphopeptide prior to incubation with the membrane (right).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 15

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.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 16

Immunohistochemical analysis using Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb on SignalSlide™ Phospho-EGF Receptor IHC Controls #8102 (paraffin-embedded KYSE450 cell pellets, untreated (left) or EGF-treated (right)).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 17

Immunohistochemical analysis of paraffin-embedded MDA-MB-468 xenograft using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (left) or PTEN (138G6) Rabbit mAb #9559 (right). Note the presence of P-Akt staining in the PTEN deficient MDA-MB-468 cells.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 18

Confocal immunofluorescent analysis of HT1080 cells, starved overnight then treated with U0126 #9903 (10 uM, 2 h; left) or PDBu (Phorbol 12,13-Dibutyrate) #12808 (100 nM, 15 m; right) using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 19

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.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 20

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

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 21

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).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 22

Immunohistochemical analysis of paraffin-embedded HCC827 xenograft, control (left) or λ phosphatase-treated (right), using Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 23

Immunohistochemical analysis of paraffin-embedded human breast carcinoma comparing SignalStain® Antibody Diluent #8112 (left) to TBST/5% normal goat serum (right) using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 24

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 25

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

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 26

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 27

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, untreated (left) or λ phosphatase-treated (right), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 28

Immunohistochemical analysis using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb on SignalSlide® Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right)).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 29

Immunohistochemical analysis using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb on SignalSlide™ Phospho-p44/42 MAPK (Thr202/Tyr204) IHC Controls #8103 (paraffin-embedded NIH/3T3 cells, treated with U0126 #9903 (left) or TPA #4174 (right).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 30

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 31

Western blot analysis of extracts from COS cells, untreated or treated with either U0126 #9903 (10 µM for 1h) or TPA #4174 (200 nM for 10 m), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 and p44/42 MAPK (Erk1/2) (3A7) Mouse mAb #9107.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 32

Immunohistochemical analysis of paraffin-embedded PTEN heterozygous mutant mouse endometrium using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb. (Tissue section courtesy of Dr. Sabina Signoretti, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.)

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 33

Western blot analysis of extracts from 293, NIH/3T3 and C6 cells, treated with λ phosphatase or TPA #4174 as indicated, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (upper), or p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695 (lower).

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 34

Immunohistochemical analysis of paraffin-embedded U-87MG xenograft, untreated (left) or lambda phosphatase-treated (right), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 35

Immunoprecipitation of phospho-Akt (Ser473) from Jurkat extracts treated with Calyculin A #9902 (100nM, 30 min). Lane 1 is 10% input, lane 2 is Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb, and lane 3 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900. Western blot analysis was performed with Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.

Phospho-EGF Receptor Pathway Antibody Sampler Kit: Image 36

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 (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

To Purchase # 9789T
製品番号 サイズ 価格 在庫
9789T
1 Kit  (8 x 20 µl)

Product 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 two western blot experiments.

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.

Source / Purification

Activation state polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Tyr239/240 of human Shc. 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, Thr202/Tyr204 of human p44 MAP kinase, or Tyr783 of human PLCγ1 protein.

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.

Pathways & Proteins

Explore pathways + proteins related to this product.

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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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