Figure 6

Recruitment of Paks to HTLV-1 LTR in the presence of Tax. (A to C) Recruitment of Pak1 (A), Pak3 (B) and Pak3-M5 (C) was assessed by ChIP. HeLa cells carrying LTR-Luc were co-transfected with expression vectors for Tax and myc-Pak1/3 or myc-M5 as indicated. After 48 h, the transfected cells were subjected to cross-linking with 1% formaldehyde for 10 min at 37°C. Cells were washed and then lysed with lysis buffer supplemented with protease inhibitor cocktail. The DNA-protein complex was sonicated, and the cell debris was removed by centrifugation. Myc-tagged Pak1/3-Tax-LTR promoter complex was immunoprecipitated using anti-myc or anti-Tax antibody and the LTR sequence was PCR-amplified. (D) Recruitment of endogenous Pak1 and Pak3. HeLa cells were co-transfected with Tax expression plasmid and pLTR-Luc. ChIP was performed with the indicated antibodies and the LTR sequence was PCR-amplified from the precipitated DNA-protein complex as above.