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. 2022 Nov 8;23(22):13742. doi: 10.3390/ijms232213742

Figure 4.

Figure 4

Tat competes against IN-CTD for TAR binding. (A) EMSA assay showing a dose-dependent competition assay of Tat on IN-CTD/TAR or IN-CTD-ΔCT/TAR complexes. TAR (50 nM), labelled with 32P (black star), was incubated with 16 fold molar excess of IN-CTD (800 nM, lanes 2–6) or IN-CTD-ΔCT (lanes 8–12) with or without increasing concentration of Tat (25, 50, 100, 200 nM). Tat vs IN-CTD is shown in lanes 3–6 and IN-CTD-ΔCT in lanes 9–12. Control binding of Tat on TAR was also performed using Tat alone (lanes 13–17). (B) Protein co-precipitation with 3′ end-biotinylated TAR (Bt-TAR). IN-CTD (lanes 1–4) or IN-CTD-ΔCT (lanes 5 and 8) were mixed with increasing amount of Tat (1, 2, 3 µg/sample; lanes 2, 3, 4 and 6, 7, 8) and incubated in a buffer containing 200 mM NaCl before co-precipitation. Tat was incubated with Bt-TAR alone (lane 9) or TAR-free beads as a control for unspecific binding (lane 10). Input (20% of total) and pull-down fractions were analyzed by 15% SDS-PAGE followed by Coomassie blue staining.