Figure 6

Effect of Tax on cellular promoters and its protein-protein interaction.A) Chromatin immunoprecipitation experiments were used to test whether Tax could occupy Sgt1 or p97 promoters. CTLL/WT and CTLL/703 (7.5 × 10⁶ cells/ChIP), as well as C81 (Tax expressing, 5.5 × 10⁶ cells/ChIP) and CEM (negative control with no Tax expression, 5.5 × 10⁶ cells/ChIP) were cross-linked, and processed for ChIP assay. Lanes 1, 4, 7 and 10 are "input" lanes where no immunoprecipitation was performed prior to PCR (positive control). Lanes 3, 6, 9 and 12 contained no antibody, and only beads, for the immunoprecipitation (negative control). Lanes 2, 5, 8 and 11 used a mixture of 500 ng of each TAb anti-Tax monoclonal antibodies (169, 170, 171 and 172; amount of each antibody judged by running 100 ng of each purified antibody on 4–20% SDS-PAGE and stained for Heavy and light chains) as the experimental sample. The high salt wash step after immunoprecipitation contained 1000 mM (as opposed to 500 mM) salt. B) Two set of C81 cells were used for immunoprecipitation followed by western blot against Tax. First, unsynchronized C81 cells (2 mg total cellular extract, Lanes 1–6) where majority of cells were at the G1 phase (71%), were used for immunoprecipitation with anti-Sgt1 (1 μg), anti-p97 (1.2 μg), anti-cdk2 (0.75 μg) and IgG (1.2 μg) followed by western blot with anti-Tax polyclonal antibody. We have previously used this method to define a list of Tax binding proteins using low and high salt wash conditions [69]. We also enriched C81 cells for G2/M population (67%) by treating with low serum (1%) and nocodazole (Noco, 50 ng/ml) for 72 hrs prior to the immunoprecipitation [15]. Lanes 4, 5, 10 and 11 served as negative control for IP, and lanes 3 and 9 served as positive control for Tax binding protein under high salt conditions (cdk2). Lanes 6 and 12 were total cellular extract (Input, 50 μg) and lanes 1, 2, 7 and 8 served as the experimental sample. The high salt wash after immunoprecipitation contained 1000 mM salt.