Analyzing Signaling Pathways Using Antibody Arrays

Cell signaling is comprised of complex networks that regulate homeostasis and human diseases. The analyses of such pathways would improve our understanding of disease pathology and direct drug development. However, it remains a great challenge to study pathways using traditional methods. We developed a high-throughput sandwich-based antibody array technology for the simultaneous detection of multiple targets, capable of identifying the relative expression levels or phosphorylation levels of major signaling pathway proteins. This array-based system features a nitrocellulose membrane or glass slide solid support, spotted with antibodies targeting key proteins of major signaling pathways, including RTK, EGFR, MAPK, AKT, apoptosis, TGFb, JAK/STAT, NFkB, and insulin receptor pathways. We employed these antibody arrays to investigate how the anti-cancer drugs, camptothecin and phorbol 12-myristate 13-acetate (PMA), alter protein phosphorylation in Jurkat and HeLa cells, respectively. Our array data suggest that camptothecin treatment induced DNA double-strand breaks in Jurkat cells and activated the DNA damage pathways ATM and Chk2, which then further induced apoptosis through caspase 3 and PARP. PMA induced the MAPK pathway in HeLa cells through the activation of ERK, CREB, and RSK1. These array results are consistent with previous studies using traditional methods and were validated with Western blotting. Our studies demonstrate that pathway antibody arrays provide a rapid, efficient, and multiplexed approach for profiling phosphorylated proteins.