Multiplexed detection of protein and phosphorylation variation in response to androgen withdrawal in LNCaP cells Free

2825

The ability to measure protein concentrations and activation states from multiple proteins in a very low volume, multiplexed format would have valuable applications in cancer studies. Here we report the development of an antibody-array platform for the parallel measurement of the abundances and phosphorylation states of numerous signaling proteins using <2 µl of whole-cell extract per assay. With two-to-four antibodies per targeted protein, sandwich assays were developed and tested efficiently for MAP kinases, AKT, GSK3B, SHIP, androgen receptor (AR), Smad family proteins, STAT5, TGFb, and HIF1A in a study of androgen-related signaling. In addition, the phosphorylation levels of those proteins were assessed using either pan-specific phospho-serine, phospho-threonine, or phospho-tyrosine antibodies, or antibodies against specific protein phosphorylation sites. Various slide blocking reagents, cell lysis buffers, and cell lysate concentrations were tested to optimize the specificity and sensitivity of the assay. Changes in the protein levels and phosphorylation states of the targeted proteins were examined in response to androgen withdrawal and di-hydrotestosterone re-stimulation of LNCaP, an androgen-dependent prostate cancer cell line. AR and Smad4 protein levels were steadily repressed after androgen withdrawal (using charcoal/dextran-treated serum), relative to control cells grown in normal serum, and AR protein levels but not Smad4 levels were restored after androgen re-stimulation. AKT protein levels dropped after androgen withdrawal and rose after about nine days, but its phospho-protein levels initially rose, then dropped (in replicate assays). Other proteins showed less dramatic but consistent androgen-related variation. These results give preliminary insights into different pathway responses to androgen withdrawal and re-stimulation and a possible shift from cell-survival activation after extended androgen withdrawal. These results also demonstrate the usefulness of miniaturized, multiplexed methods for measuring both protein and phosphorylation levels in parallel immuno-assays. [Supported by MTTC GR687 and U54DA021519]