Figure 3

INI1 binds to Tat. 293T cells were cotransfected with 5 μg of pcDNA3-Tat101-FLAG [41] and/or 5 μg of pCGN-HA-INI1 [7]. Cells were lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 4 mM EDTA, 0.1% NP-40, 10 mM NaF, 0.1 mM Na₃VO₄, 1 mM DTT and 1 mM PMSF. Lysates were pre-cleared with 30 μl of protein-G-sepharose (Amersham Biosciences). Pre-cleared supernatants were incubated with either 2 μg of anti-HA antibody (HA 11, Babco) or anti-FLAG antibody (M2, Sigma) at 4°C for 1 h. Following absorption of the precipitates on 30 μl of protein-G-sepharose resin for 1 h, the resin was washed four times with 700 μl lysis buffer. Bound proteins were eluted by boiling the resin for 5 min in 1× Laemmli sample buffer. The proteins were then subjected to SDS-PAGE, followed by immunoblotting analysis using either anti-HA or anti-FLAG antibodies.