While well suited for the high throughput needs of large-scale screens at the industrial level, such platforms may not fit the workflow or demand of smaller research groups. Such analyses require complex and expensive detection platforms such as flow cytometers or high-content imaging systems. More recently, a greater significance has been placed on the use of cell-based approaches where multiple components in a single pathway and multiple signaling cascades can be monitored simultaneously. Traditionally, compound screening was performed in cell-free assays using purified enzymes as the target. Detecting alterations in phosphorylation patterns within signaling profiles is a technique commonly employed to map the dose dependence and specificity of small-molecule inhibitors targeting upstream components. The three inhibitors (blue) are shown acting at their specific sites of signal interruption.ĭue to the inherent complexity of the global signaling network and the involvement of their constituents in malignancy, these pathways have been extensively studied by researchers looking for insight into the mechanisms underlying both normal and aberrant growth. The work performed in this study focuses on the Ras cascade (highlighted in green). Under proper conditions, phosphorylated EGFR activates any number of three downstream signaling pathways through Ras, PI3K, and JAK, respectively. The binding of EGF to the EGF receptor (EGFR) results in receptor dimerization and conformational changes triggering autophosphorylation. In fact, thirty percent of all solid tumors possess Ras or Raf mutations, including almost 90% of pancreatic adenocarcinomas. RTK mutations have been implicated in a variety of cancers, specifically, members of the epidermal growth factor receptor (EGFR) family in brain, lung, and breast cancer. The broad importance of RTK signaling is highlighted by the well-documented role of pathway dysregulation in human disease, most notably cancer. Given the dynamic interplay of cells with their surrounding microenvironment and owing to the presence of a myriad of other simultaneously activated paths, this process must be tightly regulated to ensure proper responses occur. A subsequent chain of phosphorylation events propagates the signal to the nucleus culminating in the transcription of genes required to direct changes in cell function (the EGFR cascade is outlined in Figure 1). ![]() In most instances, ligand binding initiates conformational changes in the externally facing receptor molecule leading to autophosphorylation on the internal portion of the receptor. Signaling through receptor tyrosine kinases (RTKs) is a highly conserved cellular mechanism, controlling fate determination, proliferation, survival, and migration. We report on the optimization of the assay and demonstrate its use in profiling temporal changes in phosphorylation events in the well-characterized EGF-induced signaling cascade of A431 cells. Similar in protocol to an ELISA, but based in a membrane bound 96-well microplate, the assay takes advantage of vacuum filtration to expedite the tedious process of washing in between binding steps. Capitalizing on the favorable attributes of the standard ELISA and slot blotting techniques, we developed a modified dot blot assay that provides a simple cost-effective alternative for semiquantitative expression analysis of multiple proteins across multiple samples. While offering both high-throughput and high-content analysis, these platforms are often too cost prohibitive or technically challenging for many research settings. Expression profiling on a large scale, as is the case in drug discovery, is often accomplished through use of sophisticated solid-phase protein microarrays or multiplex bead technologies.
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