Figure 1. Screening of a panel of cell lines using the PathScan® RTK Signaling Antibody Array Kit (Chemiluminescent Readout) #7982 reveals various phosphorylated RTKs and signaling nodes. A431 cells were starved for 24 hours, then treated with 100 ng/ml EGF #8916 for 3 minutes at 37ºC. NIH/3T3 cells were starved for 24 hours, then treated with 100 ng/ml PDGF #9909 for 5 minutes at 37ºC. Karpas-299 and K562 cells were lysed without starvation or treatment. The array images were captured using chemiluminescent film, with 2-5 second exposure times.
Figure 2. Treatment of MCF-7 cells with IGF-I stimulates phosphorylation of IGF-IR at tyrosine residues, Akt at Ser473 and p44/42 MAPK at Thr202/Tyr204 as detected by the PathScan® RTK Signaling Antibody Array Kit (Chemiluminescent Readout) #7982. MCF-7 cells were starved for 24 hours, then treated with 100 ng/ml IGF-I #3093 for 5 minutes at 37ºC. The chemiluminescent film image (lower panel) and the quantification of that image (upper panel) are shown. The chemiluminescent array images were captured following 2-5 second film exposures.
Figure 3. Treatment of A431 cells with EGF stimulates phosphorylation of EGFR, Akt, p44/42 MAPK and Stat3 as detected by the PathScan® RTK Signaling Antibody Array Kit (Chemiluminescent Readout) #7982. A431 cells were starved for 24 hours and treated with 100 ng/ml EGF #8916 for 5 or 40 minutes. In some cases, cells were treated with either 1 μM wortmannin #9951 for 1 hour before or 1 μM gefitinib for 2 hours before EGF stimulation. Array image pixel intensities obtained from a digital imager are shown in the top panel, while western blots are shown in the bottom panel.
Figure 4. The relationship between lysate protein concentration from untreated and IGF-I treated MCF-7 cells and the pixel intensities of phospho-IGF-IR (pan-Tyr), phospho-Akt (Ser473) and phospho-p44/42 (Thr202/Tyr204) is shown. MCF-7 cells were starved for 24 hours, then treated with 100 ng/ml IGF-I #3093 for 5 minutes at 37ºC.
Figure 5. Target map of the PathScan® RTK Signaling Antibody Array Kit (Chemiluminscent Readout)
The PathScan® RTK Signaling Antibody Array Kit (Chemiluminescent Readout) is a slide-based antibody array founded upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of 28 receptor tyrosine kinases and 11 important signaling nodes, when phosphorylated at tyrosine or other residues. Target-specific capture antibodies have been spotted in duplicate onto nitrocellulose-coated glass slides. Each kit contains two 8-pad slides, allowing the user to test up to 16 samples. Cell lysate is incubated on the slide followed by a biotinylated detection antibody cocktail. Streptavidin-conjugated HRP and LumiGLO® Reagent are then used to visualize the bound detection antibody by chemiluminescence. An image of the slide can be captured with either a digital imaging system or standard chemiluminescent film. The image can be analyzed visually or the 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).
Cell Signaling Technology's PathScan® RTK Signaling Antibody Array Kit detects the indicated RTKs and signaling nodes only when phosphorylated at tyrosine or specified residues (see Array Target Map). No significant crossreactivity has been observed between targets, with the exception of some crossreactivity of the FLT3 antibody with phosphorylated EphB3. In addition, Stat1 (Tyr701) and Stat3 (Tyr705) may be detected when phosphorylated at other tyrosine sites within the proteins. This kit is optimized for cell lysates diluted to a total protein concentration between 0.2 and 1 mg/ml (see Figure 4). All capture antibodies have been validated for human targets. Although this kit has not been tested with mouse lysates, it is expected than many capture antibodies will crossreact in murine systems.
Receptor Tyrosine Kinases (RTKs) are a family of cell surface receptors that signal primarily through tyrosine phosphorylation events (1). RTKs trigger a wide range of downstream signaling cascades, including the PI3K/Akt, MAPK and Jak/Stat pathways. These pathways control basic cellular functions such as division, growth, metabolism, differentiation, migration and survival. Dysregulation of RTK signaling has been implicated in a large number of cancers (2), making RTKs popular targets for pharmaceutical intervention.
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