Figure 1. Target map of the PathScan® EGFR Signaling Antibody Array Kit (Fluorescent Readout) #12785. † A reduction in a signal associated with E746-A750 deletion mutant was observed after treatment of cells with the small molecule inhibitors Gefitinib #4765 and Erlotinib #5083.
Figure 2. A-431 cells were grown to 90% confluency and then serum-starved overnight. Cells were stimulated with Human Epidermal Growth Factor (hEGF) #8916 (100 ng/ml, 5 min). Cell extracts were prepared and analyzed using the PathScan® EGFR Signaling Antibody Array Kit (Fluorescent Readout) #12785. Panel A shows images that were acquired using the LI-COR® Biosciences Odyssey® imaging system. Panel B shows raw values of quantified fluorescence intensity. Pixel intensity was quantified using the LI-COR® Image Studio v2.0 array analysis software.
Figure 3. Calu-3 cells were grown to 90% confluency and lysed using a buffer containing (P) or devoid of (NP) phosphatase inhibitors. Cell extracts were prepared and analyzed using the PathScan® EGFR Signaling Antibody Array Kit (Fluorescent Readout) #12785. Panel A shows images that were acquired using the LI-COR® Biosciences Odyssey® imaging system. Panel B shows raw values of quantified fluorescence intensity for a selected set of targets. Pixel intensity was quantified using the LI-COR® Image Studio v2.0 array analysis software.
Figure 4. HCC827 and H3255 are two non-small cell lung cancer (NSCLC) cell lines carrying two different gefitinib-sensitive mutants of EGFR: E746-A750 deletion in exon 19 and L858R point mutation, respectively. Cells were grown to 90% confluency and then lysed using a buffer containing (P) or devoid of (NP) phosphatase inhibitors. Cell extracts were prepared and analyzed using the PathScan® EGFR Signaling Antibody Array Kit (Fluorescent Readout) #12785. Images were acquired using the LI-COR® Biosciences Odyssey® imaging system. Fluorescence intensity was quantified using the LI-COR® Image Studio v2.0 array analysis software. Heatmap analysis was generated using MultiExperiment Viewer (MeV) analysis software (14) using the raw fluorescence intensity values.
Figure 5. (A) HCC827 cells were treated with Gefitinib #4765, Erlotinib #5083, Crizotinib #4401, or Foretinib, as indicated, for 16 hours. The total amounts of EGFR (wt and mutant) or the EGFR (E746-A750) deletion mutant were quantified using the PathScan® EGFR Signaling Antibody Array Kit (Fluorescent Readout) #12785 (bar graph). The same samples were also analyzed by western blot using EGF Receptor (E746-A750del Specific) 6B6 XP® Rabbit mAb #2085 (upper) and EGF Receptor (15F8) Rabbit mAb #4405 (lower). Note: The western blot shows that sandwich immunoassay detection in the EGFR (E746-A750) deletion mutant specific assay (bar graph) may be sensitive to EGFR inhibitors targeting the active site. (B) HCC827 cells were treated with increasing concentrations of Gefitinib #4765 for 2.5 hours. Tyrosine phosphorylation levels of the indicated sites in EGFR or the EGFR (E746-A750) deletion mutant were quantified using the PathScan® EGFR Signaling Antibody Array Kit (Fluorescent Readout) #12785.
The PathScan® EGFR Signaling Antibody Array Kit (Fluorescent Readout) uses glass slides as the planar surface and is based upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of phosphorylated EGFR, HER2, c-Met on distinct sites as well as a number of key signaling nodes found downstream of these RTKs. Target-specific capture antibodies have been spotted in duplicate onto nitrocellulose-coated glass slides. Each kit contains two slides allowing for the interrogation of 16 different samples. To improve assay performance the content of this array is split between two sub-arrays. The pads on left-hand side of each slide belong to sub-array A while the pads on the right-hand side of each slide belong to sub-array B. Cell lysates are incubated on the slide followed by a biotinylated detection antibody cocktail A or cocktail B (each cocktail for the corresponding sub-array). Streptavidin-conjugated DyLight® 680 is then used to visualize the bound detection antibody. A fluorescent image of the slide can then be captured with a digital imaging system and spot intensities quantified using array analysis software.
Kit should be stored at 4°C with the exception of Lysis Buffer, which is stored at –20°C (packaged separately).
PathScan® EGFR Signaling Antibody Array Kit (Fluorescent Readout) detects the target proteins as specified on the Array Target Map. No substantial cross-reactivity has been observed between targets. This kit is optimized for cell lysates diluted to a total protein concentration between 0.2 and 1 mg/ml (see kit protocol).
Note: Detection in the EGFR (E746-A750) deletion mutant specific assay may be sensitive to EGFR inhibitors targeting the active site. (See Figure 5).
The Epidermal Growth Factor Receptor (EGFR) is a receptor tyrosine kinase (RTK) that constitutes an important disease driver, as well as a validated drug target. The potency of EGFR in driving tumorigenesis can be attributed to its pleiotropic intracellular signaling. Activated EGFR initiates a wide range of signaling modules and switches such as the Ras - Erk/MAP kinase, Akt, Src, Stat, and PKC. Two of the most common EGFR mutations ocurring in lung cancer are the E746-A750 deletion and L858R point mutation. This array utilizes unique antibodies made by Cell Signaling Technology that are sensitive to each of these EGFR mutants, allowing specific target detection in cell extracts.
EGFR can interact and heterodimerize with other RTKs. HER2 (also known as ErbB2) is an oncogenic RTK belonging to the EGFR/HER family of RTKs and is an important heterodimerization partner of all HER family members. Another prominent heterodimerization partner of EGFR is c-Met. c-Met is an RTK serving as a receptor for the hepatocyte growth factor (HGF). c-Met can induce cell scattering, migration, and invasion. It has been shown that c-Met is responsible for some cases of tumor resistance to EGFR-targeted therapies and is a contributing factor to tumor metastasis.
PLCγ is a phosphoinositide-specific phospholipase. EGFR can activate PLCγ that, in turn, hydrolyzes phosphoinositide phospholipids residing within the inner leaflet of the plasma membrane. This hydrolysis generates two important second messengers: inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 causes calcium mobilization from intracellular storage pools, while DAG (together with calcium) activates PKC. MEK1 is a dual-specificity protein kinase and serves as the MAP kinase kinase for Erk1 and Erk2. Upon EGFR activation, MEK1 is phosphorylated by Raf and, in turn, phosphorylates the Erk kinases at Thr202 and Tyr204, leading to their activation. Activated Erk MAP kinase is a major signaling node with a multitude of substrates and primarily transmits growth and proliferation signals. Akt is another important protein kinase downstream of EGFR. Akt is activated by many RTKs and has a large number of intracellular substrates. Akt generates anabolic growth and survival signals. Stat3 is activated in response to EGFR stimulation, as well as in response to activation of a variety of cytokine receptors. Stat3 is a well-established oncogene that is also a transcription factor.
The oncogenic signals generated by activated EGFR are a focus of intense drug discovery efforts. It has become clear that in many cases a single agent inhibiting only one target is unable to cause tumor cell death in vivo. To monitor the blockade of EGFR signals alongside markers of cell death, cleaved PARP is included in this array. PARP is an enzyme involved in DNA repair. As a part of the apoptotic process, PARP is irreversibly inactivated by endoproteolytic cleavage executed by activated cell death proteases, such as caspase-3 and caspasae-7.
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