Phosphorylated STAT3 binds upstream of the human NKG2D gene translational start site. (A) The genomic sequence upstream of the NKG2D start site was analyzed using TFSEARCH software to identify putative STAT3 binding elements. Three potential regions were identified, with STAT3 binding elements indicated in underlined bold font. Sequences flanking the binding elements (identified in underlined font) were used as PCR primers. (B) Constitutive binding of pSTAT3 to the putative STAT3 binding sites was determined by PCR for each predicted region following co-ChIP with phosphorylated STAT3 from nonactivated primary human NK cells. (C) Enhanced binding of pSTAT3 to site 1 was assessed in response to treatment of NK cells with 20 ng/mL of IL10 or IL21, with or without JSI-124 for 24 hours. Data are representative of 2 replicate experiments.