LANA enhances the affinity of STAT6 bound to RTA promoter.

(A) Intact or cleaved form of STAT6 bound to RTA promoter and enhanced by LANA. HEK293 cells co-transfected with the indicated plasmids, were subjected to Chromatin immunoprecipitation (ChIP) with exogenous STAT6 (full length, Y641F, ΔTAD, ΔDBD, and ΔN). Non-specific mouse IgG and the pGL-3 reporter vector were individually used as control. The relative levels of STAT6 bound to RTA promoter were detected by quantitative PCR. Data is presented as means±SD of three independent experiments. Asterisks indicate P value as follows: *, P>0.05, and **, P<0.05. (B) Knockdown of LANA reduces the affinity of STAT6 with RTA promoter. Parental and BC3 cells with lentivirus-mediated constitutively knocked down of LANA (shLANA) or luciferase control (shCtrl) from panel A in Fig 3, were subjected to ChIP assay with endogenous STAT6 as described in panel A. (C) In vitro DNA-binding assays of intact STAT6 and its cleaved form from PEL cells. Nuclear extracts (NE) from KSHV-positive BC3 cells with or without PMSF and MG132 treatment, were individually incubated with wild-type or mutant STAT6-binding DNA oligonucleotide derived from RTA promoter followed by three washings, and the precipitates were immunoblotted with STAT6 antibody. Five percent input of DNA oligonucleotide is shown at the bottom.