Fig 3.
The APH-2 BD2 (LXXLL) motif is required for dose-dependent repression of Tax-mediated transcription and p19 Gag production. 293T cells (1.5 × 105 cells) were cotransfected with 1 μg of the wtHTLV-2 proviral clone or negative-control DNA, 0.1 μg LTR-2-Luc, 0.01 μg TK-Renilla, and various concentrations (0.2, 0.4, and 0.6 μg) of the APH, APHBD1, APHΔBD2, APHBD1ΔBD2, APHBD2ΔLL, and ΔAPH expression vectors, as indicated. (A) Tax function was measured as firefly luciferase activity from LTR-2-Luc normalized to the Renilla luciferase activity. WB analysis was used to confirm increasing concentrations of the APH-2 proteins. β-Actin levels were measured as a loading control, as shown at the bottom. RLU, relative light units. (B) The culture supernatant was collected from cells in panel A and assayed for p19 Gag production by ELISA. (C) The APH-2 BD2 (LXXLL) motif is required for interactions with CREB. IP using an anti-CREB antibody was performed on 293T cells transfected with APHΔBD2, APHBD2ΔLL, APHBD1, APH, or control expression plasmids. Immune complexes were resolved by SDS-PAGE followed by WB analysis using an APH-2-specific antiserum. Prior to IP, 10% of lysates were saved (Input) and blotted with an anti-CREB or anti-APH-2 antibody.