Phenotyping of IL4-induced nuclear Stat6 activity in humans: Quantitation after gel shift assay using immortalized cell lines

The IL4-induced Stat6 signaling pathway is active in a variety of cell types, including different cancer cells, and plays an important role in the regulation of gene expression, such as CD23. There are large quantitative differences in DNA-binding activity of IL4-induced Stat6, which are useful for phenotyping activated Stat6 in normal and disease status. However, quantitation of activated Stat6 is challenging and a standardized methodology is needed. Here we have developed a semi-quantitative methodology using gel shift assay in which IL4-induced nuclear Stat6 activities are measured in human EBV-transformed lymphoblastoid cell lines. Using a DNA probe with high affinity Stat6-binding N4 motif and a specific antibody to Stat6, autoradiographs of EMSA gels are recorded by a scan imager and OD readings of antibody super-shifted Stat6 complex bands are obtained. OD readings of all test cell lines are referenced to that of a standard cell line placed in every single experiment and an OD ratio is obtained for each test cell, which allows assignment of Stat6 activational phenotypes. Using this methodology, we have been able to define three Stat6 activational phenotypes termed as Stat6high (intense banding), Stat6low (medium intensity banding), and Stat6null (very low to no discernible banding). These Stat6 phenotypes correlate well with levels of CD23 expression, but not with those of HLA-DR. Pedigree analysis has revealed a Mendelian inheritance pattern for Stat6 phenotypes. The methodology is useful in association studies in human cancer and autoimmune diseases. The Stat6null phenotype may result from a defect in Stat6 signaling which has important implications with respect to the pathogenesis of cancer and Th1/Th2 cytokine imbalance in general. In addition, the defective Stat6null lines discovered here may serve as a natural human model for comprehensive study in the same way as a Stat6 knockout null animal model does.