Tax interacts with MCM3. (A) Lysates from HeLa cells transiently transfected with plasmids encoding either wild-type Tax (pSGTax-1) or serial deletion mutants (Δ2-6 to Δ2-55; numbers indicate Tax amino acid residue) and MCM3-Flag protein were subjected to immunoprecipitations using anti-Flag antibody (IP: α-Flag). Protein complexes were immunoblotted with anti-Flag (WB: α-Flag) or anti-Tax (WB: α-Tax) antibodies. (B-C) Tax protein was immunoprecipitated from pSGTax-1–transfected HeLa (B) or Jurkat, MT2, and C91/PL (C) cell lysates, and the protein complexes were analyzed by Western blotting using α-Tax, α-MCM2-7, or MCM3 antibodies. Arrows indicate the position of Tax-Env fusion (black arrow in MT2 cells) and wild-type Tax proteins (open arrow). Representative data of 3 independent experiments are shown. (D) HeLa cells were transfected with the pSGTax-1 vector, incubated during 16 hours, and analyzed by confocal microscopy with the indicated antibodies. In the “BrdU” panel, cells were pulsed with 33μM BrdU for 20 minutes before fixation. (E) Chromatin was prepared from JPX9 or ZnCl₂-stimulated JPX9 (JPX9i), and the presence of Tax on β-globin, Myc, LaminB2, TOP1, FMR1, and RNF185 replication origins was detected by ChIP followed by quantitative PCR analysis. Regions located 2 kb 5′ to the β-globin and Myc origins were used as control (Ctrl). (F) HeLa cells were transfected with control (Ctrl) or Tax-expression vectors (pSGTax-1 and pSGΔ2-22) and processed for ChIP experiments, as in panel E. (G) JPX9i were fixed, stained with Draq5, and sorted in G₁, early S (S_E), late S (S_L), and G₂ based on their DNA content. ChIP was performed as in panel E. (E-G) Results were expressed as a percentage of input DNA. Representative results of 1 of 3 independent experiments are shown.