Constitutive activation of Stat3 and Stat5 in HTLV-1-infected T-cell lines. (A) Western blot analysis of cellular lysates prepared from three HTLV-1-negative [HTLV-1 (-)] and three HTLV-1-infected [HTLV-1 (+)] T-cell lines. The blots were probed with anti-phospho-Stat3, anti-Stat3, anti-phospho-Stat5, anti-Stat5 and anti-Tax. Amounts of actin are shown as loading controls. (B) Stat-DNA binding activities in HTLV-1-negative and HTLV-1-infected T-cell lines were detected by EMSA using SIE or β-casein probe. Arrows indicate specific protein-DNA complexes. NS indicates non-specific bands. (C) Competition assay was performed with nuclear extracts of HTLV-1-infected cell lines using 100-fold excess of unlabeled wild type (W) or mutant (M) oligonucleotide as a competitor (upper panels: SIE, lower panels: β-casein). (D and E) Involvement of Stat3 and Stat5 in the formation of SIE- (D) and β-casein- (E) binding complexes in HTLV-1-infected T-cell lines. EMSA was performed with nuclear extracts of the indicated cell lines either in the absence (-) or presence of a specific Stat antibody (αStat: anti-Stat1, Stat2, Stat3, Stat4 and Stat5 antibodies). The supershifted complexes are indicated by arrowheads.