Figure 1: Measurement of phosphorylated and total STAT.
NIH/3T3 cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 50 ng/ml PDGF for 5 minutes and fixed. Total and phospho STAT2, STAT4 and STAT6 were each assayed in triplicate using the phospho and total STAT antibodies included in the FACE STAT Kits. Data was plotted after correction for cell number (performed through use of Crystal Violet).
The phospho-STAT2 antibody is specific for phosphorylated STAT2 at Tyrosine 689 and does not cross-react with other sites. The total-STAT2 antibody recognizes STAT2 proteins regardless of the phosphorylation state. The phospho-STAT4 antibody is specific for phosphorylated STAT4 at Tyrosine 693 and does not cross-react with other sites. The total-STAT4 antibody recognizes STAT4 proteins regardless of the phosphorylation state. The phospho-STAT6 antibody is specific for phosphorylated STAT6 at Tyrosine 641 and does not cross-react with other sites. The total-STAT6 antibody recognizes STAT6 proteins regardless of the phosphorylation state.
Signal transducers and activators of transcription (STAT) proteins are latent transcription factors that are activated by phosphorylation via tyrosine kinases. Over 35 different extracellular polypeptides activate Janus kinase associated receptors, leading to phosphorylation of Janus kinases and the subsequent phosphorylation of STAT proteins. Upon phosphorylation, the STAT proteins dimerize and migrate to the nucleus where they exert transcriptional activation. Phosphorylation of a single tyrosine localized around residue 700 is crucial for activation of each STAT family member. STAT1 is involved in the activation of IFN? and IFN? genes, STAT2 in the activation of IFN? genes, STAT4 and STAT6 in T-helper