Multiplexed Transcription Factor Probes
Each TF probe contains a double-stranded protein-binding region to which a biotin label is attached. Multiple TF probes, each with a different protein-binding sequence, are mixed with a nuclear extract, and the TFs are allowed to bind to the DNA. Once binding has occurred, a proprietary reagent is added that removes the biotin from the binding sites that have not been protected by bound TFs from the nuclear extract. A summary of the protocol is shown in Figure 1.
Next, the probes are hybridized to a set of spectrally distinguishable microspheres containing covalently attached single-stranded DNA oligonucleotides that are complementary to corresponding capture sequences included in each TF probe. Biotin molecules remaining on probes containing bound, activated TF complexes are detected with a streptavidin-phycoerythrin conjugate, and the signals are measured in a Luminex 100 instrument (www.luminexcorp.com).
Anna Lokshin, Ph.D., director of the Luminex Core Facility at the University of Pittsburgh, has been using Marligens multiplex TF assay for some time. One of Dr. Lokshins ongoing projects has been a study of the mechanism and pathways involved in the stimulation of dendritic cells, which are involved in the presentation of antigens to nave T cells in the primary immune response.
The importance of antigen presentation in the generation of specific immune response has been confirmed in vivo by the demonstration that blocking antigen presentation down-regulates both humoral and cell-mediated immune responses, including antitumor immunity. Dendritic cell function is regulated by maturation state, and appropriate differentiation and maturation is a crucial factor for a fully functional immune system.
In Dr. Lokshins studies, human dendritic cells were generated from CD14+ PBMC in cultures supplemented with GM-CSF and IL-4 for 7 days. To induce activation of NF-kB, dendritic cells were treated with TNF-a (50 ng/mL) for 15 and 30 minutes. Non-treated dendritic cells served as a control.
Nuclear extracts were prepared and tested with Marligens 10-plex TF profiling assay. The data shown (see Figure 2), provides a dynamic picture of TF activation in TNF-a-treated human dendritic cells. A peak of NF-kB activity was observed at 15 minutes, in agreement with previously published data. More importantly, however, a strong time-dependent activation of EGR, NF1, and PPAR was also observed in TNF-a-treated dendritic cells. The involvement of these TFs in dendritic cells activation by TNF-a has not been previously described.
These findings allow the exploration of the role of these TFs in dendritic cell maturation, activation and function, demonstrating the utility of multiplexed TF screening assays in biological research.