FIG. 4.

LANA suppresses IL-4-stimulated activation of STAT6 by affecting its DNA-binding ability. (A) LANA reduces the IL-4-induced phosphorylation of exogenous STAT6. DG75 cells were transiently transfected with FLAG-STAT6 (10 μg) or with LANA-myc (0, 5, 10, 15, or 20 μg). The equal amounts of DNA were made up with empty vector. Forty-eight hours posttransfection, cells were treated with 5 ng/ml IL-4 for 2 h before harvest. Whole-cell lysates (WCL) were subjected to immunoprecipitation (IP) with anti-FLAG followed by immunoblotting (IB) against p-STAT6. The membrane was stripped and reimmunoblotted (reIB) for anti-FLAG, or immunoblotting was directly performed with anti-myc as indicated on the figure. (B) Luciferase (Luc) reporter assays of IL-4-induced transcriptional activity of phosphorylated STAT6. BJAB cells with LANA (YLF) or GFP (Vector) stable expression, or with STAT6 knockdown (shSTAT6) or scramble control (shCtrl) generated by lentivirus-mediated transduction followed by 2 μg/ml puromycin selection (Western analysis was performed to detect LANA expression and STAT6 knockdown efficiency), were individually transfected with FLAG-STAT6 (0, 10 μg) in the presence of the reporter 3×stat6-luc (10 μg). At 48 h posttransfection, cells were treated or not treated with IL-4 for 2 h before harvest. Cell lysates were performed reporter assays. The results are presented as the fold change compared with vector alone. RLU, relative luciferase units. Asterisks indicate the fold change after IL-4 induction. (C) In vitro DNA-binding assays of phosphorylated STAT6 in the presence of LANA. Whole-cell extracts with preadjusted equally IL-4-induced phosphorylated STAT6 from BJAB/vector or BJAB/YLF were individually incubated with wild-type or mutant STAT6-binding DNA oligonucleotide followed by three washings, and the precipitates were Western blotted with p-STAT6 antibody. Five percent input of DNA oligonucleotide is shown at the bottom. RD, relative density.