(A) U87MG cells were transfected with Flag-TRAF3, and cell lysates were incubated with an anti–Flag-Tag antibody. The immunoprecipitated protein complexes were resolved by SDS-PAGE, and specific protein bands, identified by silver staining, were subjected to MS analysis. The black arrowhead indicates the TRAF3 band, and the white arrowhead indicates the ECH1 band identified by tandem MS/MS. IgH and IgL indicate the IgG heavy chain and light chain, respectively. H, heavy chain; L, light chain. (B) Coimmunoprecipitation assays demonstrate the reciprocal interaction between TRAF3 and ECH1 in GBM0709 and GBM0108 cells after treatment with 5-Aza. (C) Myc-tagged full-length ECH1 or deletion mutants were cotransfected with Flag-TRAF3 into HEK293T cells. Cell lysates were immunoprecipitated using an anti-Myc antibody, and the immunoprecipitates were analyzed by immunoblotting (IB) using the indicated antibodies. (D) HEK293T cells were transfected with the Myc-ECH1, Flag-TRAF3, and HA-Ub plasmids. The cell lysates were incubated with an anti–Myc-tag antibody and then subjected to immunoblotting. (E) GBM0108 cells were transfected with TRAF3 siRNA and HA-ubiquitin (HA-Ub), and then were treated with 5-Aza. The cell lysates were immunoprecipitated with an anti-Myc antibody and then analyzed by immunoblotting. (F) HEK293T cells were transfected with the indicated plasmids and then incubated with an anti–Myc-tag antibody. The resultant immunoprecipitates were analyzed by immunoblotting. (G) The binding mode between TRAF3 and ECH1 was predicted by the HDOCK server. The protein tertiary structures for TRAF3 and ECH1 were retrieved from the Alphafold database. Yellow dashed lines indicate possible interactions, and residues highlighted in red indicate potential residues mediating protein-protein interactions. The interatomic distances between TRAF3 and ECH1 are indicated. (H) HEK293T cells were transfected with Flag-TRAF3, HA-Ub-K63, and Myc-ECH1 or each of the ECH1 mutants (K214R or K276R). The cell lysates were immunoprecipitated with an anti-Myc antibody and then analyzed by immunoblotting. (I) In vitro ubiquitination assay using the indicated purified proteins in the presence of E1 and E2 enzymes and Mg²⁺-ATP (10 mM) in the ubiquitination reaction buffer. The reaction mixtures were purified by Ni-NTA beads, and the eluted proteins were analyzed by immunoblotting. M, molecular weight; K, kDa.