Figure 6. Timeless downregulation reduces the level of replisome association at telomeres. (A) Western analysis of cells used in ChIP experiments. (B) HeLa cells expressing the indicated shRNA (C: Control; T: Timeless) were transfected with control vector (Vec), pcDNA3-TRF1-FL (TRF1). Cells were processed for ChIP using antibodies against Cdc45 or RPA. Precipitated DNA recovered from antibody-conjugated beads was used to monitor the association of Cdc45 or RPA using primers targeting different sub-telomeric (TTAGGG)_n repeats (ST1 and ST2). TRF1 overexpression results in an increase in replisome association at sub-telomeres, which was abrogated by Timeless depletion. Relative DNA precipitation from each sample to control precipitation (set to 1) is shown for each experiment. Data are from at least three independent experiments, and error bars represent standard deviations. (C) Cell cycle analysis of cells used in B was performed using flow cytometry.