Fig. 3.

A lysine-less TRIM21^RING-Box autoubiquitinates with Ube2W and Ube2N/Ube2V2 in vitro. (A–C) WT or mutant TRIM21^RING-Box-6KR proteins bearing lysine-to-arginine at residues 45, 61, 77, 105, 108, and 119 were incubated with E1 and Ub in the presence or absence of ATP and Ube2W (A), Ube2N/Ube2V2 (B), or Ube2W and Ube2N/Ube2V2 (C). Reactions products resolved by LDS-PAGE and visualized by Coomassie stain. (D and E) Autoubiquitination of WT (D) or 6KR (E) TRIM21^RING-Box in the presence of Ube2W (Left) or Ube2W and Ube2N/Ube2V2 (Right). Reaction products resolved by LDS-PAGE and visualized by immunoblot detecting TRIM21. Reactions containing TRIM21^RING-Box in the absence of any E2 served as controls.