LANA promotes nuclear localization of STAT6 independent of Y641-phosphorylation.

<p>(<b>A</b>) Inhibition of LANA blocks nuclear localization of STAT6. Confocal microscopy of endogenous STAT6 (red) and merged in PEL cells BC3 with lentivirus-mediated constitutively knocked down of LANA (shLANA) or luciferase control (shCtrl). Nuclei were stained with DAPI. (<b>B</b>) Western blot analyses of fractionated BC3 cells with LANA or luciferase control knockdown. Cyt, cytosolic; Nuc, nuclear; were revealed by α-tubulin and Histone H3, respectively. (<b>C</b>) LANA induces nuclear localization of exogenous STAT6 independent of phosphorylation on tyrosine 641. HEK293 cells transfected with wild type (WT) or Y641 mutant (Y641F) of STAT6 with FLAG tag (green) in the presence or absence of LANA with RFP tag (red) were imaged by confocal assay. Nuclei were stained with DAPI. The relative percentage of STAT6 nuclear localization (right panel) was individually quantitated by nuclear and cytoplasmic staining of 100 cells. (<b>D</b>) Immunoblotting analyses of fractionated 293 cells transfected with expressing plasmids as indicated. Cyt, cytosolic; Nuc, nuclear; were revealed by α-tubulin and Histone H3, respectively. (<b>E</b>) Cells from panel C in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006124#ppat.1006124.g001" target="_blank">Fig 1</a> or panel D were individually treated with DSS for 30 minutes before lysis and analyzed by immunoblotting (IB) for dimerization of STAT6 (data is shown on the left and right panels, respectively). Cells treated with IL-4 (50 ng/ml) for 30 min were used as control.