Figure 5

Competition of TAR/TAR complex formation by NF90c protein. One microgram of purified biotin-labeled TAR RNA was mixed with one microgram of purified Tat protein for 10 minutes on ice. Next, 100 μl of 30% strepavidin sepharose beads in binding buffer (50 mM Tris-HCl, pH7.8; 5 mM DTT, 100 μg of BSA, 60 mM KCl and 5 mM MgCl₂) were added to the reaction for a final volume of 200 μl. The TAR/Tat complex was incubated with beads for an additional 1 hr on ice. Next, purified NF90c at various concentrations (0.1, 1, and 5 μg) were added to the mixture. All samples were further incubated on ice for additional one hour. Finally, samples were centrifuged at 4°c for 5 minutes, and washed (3×) with TNE₃₀₀ + 0.1% NP-40 (50 mM Tris-HCl, pH7.8, 300 mM NaCl, 1 mM ETDA, plus 0.1% NP-40). A final wash was with TNE₅₀ + 0.1% NP-40 was performed. Bound complexes were separated on a 4–20% SDS/PAGE and western blotted with anti-Tat mAb or anti-NF90c antibodies. Same Blot was cut in half for either Tat or NF90c western blot. Lane 1: ¹⁴C protein molecular weight marker, Lane 2 is with no Tat, Lane 3 with Tat, Lane 4 and 5 are with addition of five microgram of either wild type TAR or a mutant TAR RNA (TM26) as specific and non-specific competitors, respectively. Lanes 6–8 represent addition of purified NF90ctv protein in presence of constant amount of Tat.