Revision 1

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Support:

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3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Mol. Wt Isotype/Source
Phospho-EGF Receptor (Tyr1068) (D7A5) Rabbit Monoclonal Antibody377720 µl175 kDaRabbit IgG
Phospho-Akt (Ser473) (D9E) Rabbit Monoclonal Antibody406020 µl60 kDaRabbit IgG
Phospho-Gab1 (Tyr627) (C32H2) Rabbit Monoclonal Antibody323320 µl110 kDaRabbit IgG
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit Monoclonal Antibody437020 µl44, 42 kDaRabbit IgG
Phospho-Shc (Tyr239/240) Antibody243420 µl50, 55, 70 kDaRabbit
Phospho-Stat5 (Tyr694) (D47E7) Rabbit Monoclonal Antibody432220 µl90 kDaRabbit IgG
Phospho-c-Cbl (Tyr700) (D16D7) Rabbit Monoclonal Antibody886920 µl120 kDaRabbit IgG
Phospho-PLC gamma1 (Tyr783) (D6M9S) Rabbit Monoclonal Antibody1400820 µl155 kDaRabbit IgG
Anti-rabbit IgG, HRP-linked Antibody7074100 µlGoat

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

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.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

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).

Trademarks and Patents

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.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
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).
undefined Image 1: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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).
undefined Image 2: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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).
undefined Image 3: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

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Western blot analysis of extracts from serum-starved A431 cells, untreated (-) or treated with hEGF (100 ng/ml, 5 min; +), using EGF Receptor (1F4) Mouse mAb #2239 (Panel A) and Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb #3777 (Panel B). Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) #5151 (green) and Anti-mouse IgG (H+L) (DyLight 680 Conjugate) #5470 (red) were used as secondary antibodies.
undefined Image 4: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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).
undefined Image 5: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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.
undefined Image 6: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

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Immunohistochemical analysis of paraffin-embedded human urothelial carcinoma using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb performed on the Leica BOND RX.
undefined Image 7: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Western blot analysis of extracts from various cell lines treated with TPA (200nM, 4 hr) or PDGF (100ng/mL, 10 min) as indicated, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (upper), p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
undefined Image 8: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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.
undefined Image 9: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

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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).
undefined Image 10: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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.
undefined Image 11: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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).
undefined Image 12: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

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Immunohistochemical analysis of paraffin-embedded HCC827 xenograft, control (left) or λ phosphatase-treated (right), using Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb.
undefined Image 13: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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.
undefined Image 14: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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).
undefined Image 15: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

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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).
undefined Image 16: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human squamous cell carcinoma of the skin using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb performed on the Leica BOND RX.
undefined Image 17: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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).
undefined Image 18: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

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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)).
undefined Image 19: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Simple Western™ analysis of lysates (0.1 mg/mL) from A431 cells treated with human EGF (100 ng/mL, 30”) using Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb #3777. The virtual lane view (left) shows a single target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
undefined Image 20: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.
undefined Image 21: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

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Flow cytometric analysis of TF-1 cells, untreated (blue, low) or treated with hGM-CSF #8922 (50 ng/ml, 15 min; green, positive) using Phospho-Stat5 (Tyr694) (D47E7) 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.
undefined Image 22: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb.
undefined Image 23: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb performed on the Leica BOND RX.
undefined Image 24: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

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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.
undefined Image 25: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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.
undefined Image 26: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.
undefined Image 27: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

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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.
undefined Image 28: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb performed on the Leica BOND RX.
undefined Image 29: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Confocal immunofluorescent analysis of HeLa cells, untreated (left) or EGF-treated (right), using Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb (green) and DRAQ5® #4084 (blue pseudocolor).
undefined Image 30: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

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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.)
undefined Image 31: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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).
undefined Image 32: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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).
undefined Image 33: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

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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.
undefined Image 34: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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).
undefined Image 35: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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.
undefined Image 36: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

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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).
undefined Image 37: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb.
undefined Image 38: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded U-87MG xenograft, untreated (left) or lambda phosphatase-treated (right), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.
undefined Image 39: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

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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.
undefined Image 40: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Immunohistochemical analysis of paraffin-embedded 4T1 syngeneic tumor using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb.
undefined Image 41: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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)).
undefined Image 42: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

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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).
undefined Image 43: Phospho-EGF Receptor Pathway Antibody Sampler Kit
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.
undefined Image 44: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Immunoprecipitation of Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) from 3T3 cell extracts. Cells were treated with TPA, (200 nM, 15 min). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb. Western blot was performed using Phosphop44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Light-Chain Specific) (D4W3E) mAb #45262 was used as a secondary antibody.
undefined Image 45: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

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Simple Western™ analysis of lysates (0.1 mg/mL) from serum-starved A431 cells treated with hEGF (100 ng/mL, 5 min.) using Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb #14008. The virtual lane view (left) shows a single target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
undefined Image 46: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Simple Western™ analysis of lysates (0.1 mg/mL) from Jurkat cells treated with Calyculin A (100 uM, 30 min) using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
undefined Image 47: Phospho-EGF Receptor Pathway Antibody Sampler Kit

Western blot analysis of extracts from various cell lines, untreated (-) or treated (+) as indicated with human insulin (100 nM, 20 min), mouse PDGF-BB (100 ng/ml, 20 min), or human Insulin-like Growth Factor I (hIGF-I) (100 ng/ml; 5 min), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

undefined Image 48: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

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Western blot analysis of extracts from MCF-7 cells, untreated (-) or treated with hIGF-1 (100 ng/ml, 10 min; +), using Akt (pan) (E7J2C) Mouse mAb #58295 (Panel A) and Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (Panel B). Anti-mouse IgG (H+L) (DyLight 680 Conjugate) #5470 (red) and Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) #5151 (green) were used as secondary antibodies.
undefined Image 49: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Simple Western™ analysis of lysates (1 mg/mL) from serum-starved NK-92 cells treated with hIL-2 (10 ng/mL, O/N) using Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb #4322. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
undefined Image 50: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Simple Western™ analysis of lysates (0.1 mg/mL) from serum-starved HeLa cells treated with TPA (400 nM, 4 hours) using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370. The virtual lane view (left) shows the target bands (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
undefined Image 51: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

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Western blot analysis of extracts from Jurkat cells, treated with U0126 (10 µM, 1 hour) (negative control) or treated with TPA (200 nM, 20 min) (positive control), using p44/42 MAPK (Erk1/2) (L34F12) Mouse mAb #4696 (Panel A) and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (Panel B). Anti-mouse IgG (H+L) (DyLight 680 Conjugate) #5470 (red) and Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) #5151 (green) were used as secondary antibodies.
undefined Image 52: Phospho-EGF Receptor Pathway Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.