Figure 4. Timeless interacts with TRF1 and TRF2. (A) Timeless associates with TRF1 and TRF2. 293T cells were transfected with the indicated plasmids to express Timeless or Tipin. 48 h later, immunoprecipitation (FLAG-IP) was performed on cell extracts with the anti-FLAG beads. Associated proteins were probed with anti-TRF1 and anti-TRF2 antibodies. (B) 293T cells were transfected with indicated plasmids to express Shelterin components. 48 h later, immunoprecipitation (FLAG-IP) was performed using anti-FLAG beads. Immunoprecipitates were probed with indicated antibodies to determine interaction between Timeless and Shelterin components. (C) The indicated GST-fused recombinant proteins were incubated with the Timeless protein for 45 min. GST-fusion proteins were precipitated using glutathione Sepharose beads and analyzed by western blotting using the anti-Timeless antibody (Timeless WB). Recombinant Timeless and GST-fusion proteins were stained by Colloidal blue staining. WCE, whole cell extract; FLAG-IP, immunoprecipitated fraction; WB, western blotting. Representative results of repeat experiments are shown.