Rapid Screening of the Epidermal Growth Factor Receptor Phosphosignaling Pathway via Microplate-Based Dot Blot Assays

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. While offering both high-throughput and high-content analysis, these platforms are often too cost prohibitive or technically challenging for many research settings. 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. 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. 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. 1. Introduction Signaling through receptor tyrosine kinases (RTKs) is a highly conserved cellular mechanism, controlling fate determination, proliferation, survival, and migration [1, 2]. In most instances, ligand binding initiates conformational changes in the externally facing receptor molecule leading to autophosphorylation on the internal portion of the receptor. 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). 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. The broad importance of RTK signaling is highlighted by the well-documented role of pathway dysregulation in human disease, most notably cancer. 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. In fact, thirty percent of all solid tumors possess Ras or Raf mutations, including almost 90% of pancreatic adenocarcinomas [3, 4]. Figure 1: The EGFR signaling cascade. The binding of EGF to the EGF receptor (EGFR) results in receptor dimerization and conformational changes triggering autophosphorylation. Under proper conditions, phosphorylated EGFR activates any number of three downstream signaling pathways through Ras, PI3K, and JAK, respectively.