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. 2015 Jul 6;112(32):10014–10019. doi: 10.1073/pnas.1507534112

Fig. 2.

Fig. 2.

TRIM21 autoubiquitinates with Ube2W and Ube2N/Ube2V2 in vitro. (A and B) Ube2W monoubiquitinates TRIM21 (T21). Purified MBP-T21 (A) or T21RING-Box (RB) (B), E1, Ub, and ATP were incubated with increasing concentrations of Ube2W. Reaction products separated by LDS-PAGE and visualized by IB detecting T21 (Top) or Ub (Bottom). (C and D) Ube2W anchors polyUb to T21. Ubiquitination reactions as in B, but with increasing concentrations of Ube2N/Ube2V2 (2N/2V2) in the absence (C) or presence (D) of Ube2W (2W). (E) T21(RB) autoubiquitinates with Ub-63Ub. T21(RB) incubated with E1, ATP, 2W, 2N/2V2, and WT Ub or Ub bearing lysine-to-arginine substitutions at residue 63 (K63R), residue 48 (K48R), or at all Ub lysine residues except residue 63 (K63 only).