Development of cell-based assays for cytokine receptor signaling, using an AlphaScreen SureFire assay format.
The signal transducers and activators of transcription (STAT) proteins are a small family of signaling proteins that are crucial for cytokine and growth factor receptor-mediated signaling in various blood cell types. Despite their central role in immune and hematopoietic cellular regulation, there are relatively few options for monitoring receptor-mediated JAK/STAT signaling events in a cell-based format, without the need for cellular transfections or labor intensive methodology. Indeed, traditional methods such as the Western blot or ELISA remain a standard method for determining the phosphorylation status of endogenous STAT proteins. Here we present data for the rapid detection of endogenous receptor-mediated phosphorylation of multiple STAT proteins using the bead-based AlphaScreen SureFire technology. With three different cell lines (human acute monocytic leukemia THP-1 cells, human erythroleukemic TF-1 cells, and human T lymphocytic Jurkat cells), we have optimized a rapid and homogeneous methodology for monitoring endogenous, receptor-mediated signaling via STAT 1, STAT 3, or STAT 5 phosphorylation, in response to several agonists. These assays, which can be tailored for both standard research applications or high-throughput drug screening applications, afford quantitative data for receptor-mediated signaling mechanisms in an endogenous, cellular environment.
Osmond
Ronald Ian William
RI
TGR BioSciences, Thebarton, South Australia 5031, Australia. ron.osmond@tgr-biosciences.com.au
Das
Subhobrata
S
Crouch
Michael Francis
MF
D001671:Biochemistry / Q000379:methods; D015153:Blotting, Western; D045744:Cell Line, Tumor; D006801:Humans; D019169:Jurkat Cells; D010766:Phosphorylation; D018121:Receptors, Cytokine / Q000378:metabolism*; D050791:STAT Transcription Factors / Q000378:metabolism*; D050794:STAT1 Transcription Factor / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction
D018121:Receptors, Cytokine; D050791:STAT Transcription Factors; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor