Fig. 1

UBE2O combines with PTRF directly. A PTRF co-immunoprecipitates UBE2O. Myc-UBE2O and Flag-PTRF expression plasmids were co-transfected into HEK293T cells by using PEI transfection. Cell lysates were prepared after 40 h transfection, and Flag-PTRF in the cell lysates combined with FLAG M2 beads. The immunoprecipitates and the total cell lysates were analyzed by immunoblotting (IB) as shown. B Endogenous PTRF in HeLa cells was immunoprecipitated with PTRF antibody by protein A. PTRF-associated endogenous UBE2O was detected by IB. C Wild-type PTRF and PTRF deletion mutants D1, D2 and D3 were shown as schematic representations. D UBE2O-Myc and Flag-PTRF or Flag-PTRF deletion constructs (D1–D3) vectors were transfected into HEK293T cells. Flag M2 beads immunoprecipitates and the total cell lysates were immunoblotted with Myc and Flag antibodies. E Wild-type UBE2O and UBE2O deletion mutants D1, D2, D3, D4 and D5 were shown as schematic representations. F Myc-PTRF and Flag-UBE2O or five UBE2O deletion vectors (D1–D5) were transfected into HEK293T cells. Cell lysates were analyzed by IP and IB as indicated. G GST and GST-PTRF D3 were purified on GST-agarose, and His-UBE2O CR2 was purified on Ni-agarose. The framed strips were the purified protein strips. H The purified GST or GST-PTRF D3 was applied to the purified His-UBE2O CR2 immobilized on Ni–NTA superflow as indicated. Eluted proteins were analyzed by western blotting