Pcdna3 vector

Subtype B TAR was cloned between HindIII and BamHI site in pCDNA3 vector (Promega). ³²P-labeled TAR was transcribed in vitro using T7 RNA polymerase. TAR was incubated with increasing amounts of purified Tat protein (0.1–2 μg) for 10 min on ice, followed by 10 min at 37°C with binding buffer (Promega). The reaction was stopped by adding 4X gel loading buffer and Tat variants with TAR complexes were analyzed on 4% Non-denaturing polyacrylamide gels and autoradiography was done. 1 μg of the empty pCMV-myc empty vector was used as a control and the expression of Tat–TAR binding was normalized with interaction with empty vector; the experiment was repeated three times.

Recombinant SOX17-Flag and Flag alone (pcDNA3 vector (Promega)) were expressed in 293T cells. Plasmids were transfected using Lipofectamine 2000 Transfection Reagent (Life Technologies, 11668019) 36 h before cells were lysed in RIPA buffer containing protease inhibitors. Recombinant protein was immunoprecipitated from lysate overnight at 4 °C with Anti-FLAG M2 magnetic beads (Sigma, M8823) and the recombinant protein eluted with excess FLAG peptide. 5–7 μl of the first eluate was used in a binding reaction along with 0.3 pmol of complementary annealed 3′ Biotin-labelled oligonucleotides (Integrated DNA Technologies), 300-fold excess competitor probes, 0.02U Poly(dG–dC) (Sigma, P9389) and binding buffer (100 mM HEPES pH 8.0, 50 mM KCl, 500 μM dithiothreitol, 50 μM EDTA, 1 mM MgCl₂ and 5% glycerol by volume)66 (link). DNA–protein complexes were resolved on 7% native polyacrylamide gel, transferred to neutrally charged nylon membrane, incubated with Streptavidin-POD (Roche, 11089153001) and imaged by chemiluminescence. See Supplementary Table 4 for probe sequences.