APH-2 represses Tax2-dependent activation of transcription from the HTLV-2 LTR. (A) The 293T cells were transiently transfected with 125 ng pHTLV-2-LTR-luc (left) or pHTLV-1 LTR-luc (right), 500 ng pSG5M-Tax2 (left), or pSG5M-Tax1 (right) together with increasing amounts (125, 250, 500, and 1000 ng in both cases) of APH-2-Myc (left) or of HBZ-SP1-Myc (right). The graphs represent an average of 3 (left) or 2 (right) independent experiments. (B) A total of 50 μg protein extracts from lysates obtained from transfected 293T cells were subjected to electrophoresis and probed for Myc, Tax2, Tax1, and β-tubulin. (C) The 293T cells were transiently transfected with 250 ng pH6neo, 62.5 ng pHTLV-2-LTR-luc, 50 ng pRcActin-LacZ together with increasing amounts (0, 62.5, 125, 250, and 500 ng) of the APH-2-Myc vector. Cells were lysed 48 hours after transfection, and luciferase activities were measured using the MLX microplate luminometer (Dynex Technologies). The β-galactosidase activity was measured using the Galacto-Light kit (Applied Biosystems) according to the manufacturer's suggestions. Luciferase activities are presented as normalized relative light units (RLU/β-gal) and correspond to the calculated mean ±SD of 3 transfected samples normalized by the measured β-galactosidase activity. (D) The 293T cells were transiently transfected with 2.5 μg pH6neo, increasing amounts (0, 500, 1000, 2000, and 4000 ng) of the APH-2-Myc vector. Cell lysates were subjected to electrophoresis and probed for p24 or GAPDH. (E) For detection of p19 antigen in supernatants, 293T cells were transiently transfected with 250 ng pH6neo with increasing amounts (0, 62.5, 125, 250, and 500 ng) of APH-2-Myc vector. Cell supernatants were used for p19 enzyme-linked immunosorbent assay according to the manufacturer's instructions (HTLV-I/II p19 Antigen ELISA; ZeptoMetrix Corporation). Experiments were done in triplicate, and values are representative of the calculated mean ± SD.