FIG. 6.
Tat displaces SATB1-bound HDAC1 in vitro. (A) A competition assay was performed to analyze the binding of HDAC1 and Tat to SATB1 in vitro. The SATB1-HDAC1 complex was isolated using anti-FLAG affinity beads in the FLAG pulldown assay as described in Materials and Methods (lane 1). The complex was incubated with 2 and 4 μg of recombinant GST-Tat and pulled down using FLAG beads (lanes 2 and 3, respectively). Lane 4 represents the input lysate. As a control, the complex was incubated with 2 and 10 μg of purified GST and pulled down with FLAG beads (lanes 5 and 6). The lower panel represents an immunoblot analysis of these samples using anti-HDAC1. (B) A competition assay was performed to analyze the HDAC1-displacing ability of mutant and truncated Tat from the HDAC1-SATB1 complex in vitro. 293 cells were transiently transfected with FLAG-HDAC1, and the SATB1-HDAC1 complex was isolated using anti-FLAG affinity beads in the FLAG pulldown assay. The complex was incubated with 2 and 4 μg of recombinant wild-type GST-Tat (lanes 1 and 2), GST-Tat 20-72 (lanes 3 and 4), GST-Tat 30-72 (lanes 7 and 8), and GST-Tat 40-72 (lanes 9 and 10, respectively) and pulled down using FLAG beads. In the case of pulldown using the GST-Tat C22G mutant, 2 and 8 μg of protein were used (lanes 5 and 6, respectively). The middle panel represents the purified GST-Tat proteins stained with Coomassie brilliant blue. The lower panel represents an immunoblot analysis of these samples using anti-HDAC1 and serves as loading control.