Figure 1. Screening of cell lines using the PathScan® RTK Signaling Antibody Array (Fluorescent Readout) #7949 reveals various phosphorylated RTKs and signaling nodes. Karpas-299 and K562 cells were lysed without starvation or treatment. The fluorescent image (lower panel) and the quantification of that image (upper panel) are shown.
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 (Fluorescent Readout) #7949. MCF-7 cells were starved for 24 hours, then treated with 100 ng/ml IGF-I #3093 for 5 minutes at 37ºC. The fluorescent image (lower panel) and the quantification of that image (upper panel) are shown.
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 (Fluorescent Readout) #7949. A431 cells were starved for 24 hours and treated with 100 ng/ml hEGF #8916 for 5 or 40 minutes. In some cases, cells were treated with either 1 μM wortmannin #9951 for 1 hour or 1 μM gefitinib for 2 hours before EGF stimulation. Fluorescence intensities obtained from the array 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 relative fluorescence of phospho-IGF-IR (panTyr), 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 (Fluorescent Readout)
The PathScan® RTK Signaling Antibody Array Kit (Fluorescent Readout) is a slide-based antibody array product 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 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).
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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