Figure 4

³²P labeled RNA was made using T7 RNA polymerase after linearizing the vector as described by the manufacturer (Promega) and as described earlier [19]. Increasing amounts of purified GST-Rev B or C (0.1 to 0.7 μM) was mixed with fixed amounts of labeled RRE in 10 μl of binding buffer (10 mM HEPES/KOH pH 7.6, 150 mM KCl, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM DTT, 20% (V/V) glycerol, 3.2 μg E. coli t-RNA) and subjected to EMSA under exactly identical conditions as described before [18].