Fig. 1: Acetylated Ub variants and their performance in HDM2 autoubiquitylation.

a Lysine residues of ubiquitin (Ub) used for Ub chain formation and acetylation (Ac) in cells. b, c Ub variants acetylated at K6 (6AcK), K11 (11AcK), K27 (27AcK), or K48 (48AcK) are inefficiently used by HDM2 for autoubiquitylation. d Autoubiquitylation assays with Ub variants, in which K48 was replaced by A, Q, or R reveal that the presence of a positive charge at position 48 is critical for efficient HDM2 autoubiquitylation. e Autoubiquitylation assays with Ub variants, in which K11 was replaced by A, Q, or R, indicate that the inefficient use of Ub 11AcK by HDM2 for autoubiquitylation cannot be explained by charge neutralization only. b–e Autoubiquitylation reactions were performed as described in Methods and started by addition of the respective Ub variants or nonmodified Ub (wt). Reactions were stopped at the times indicated. Reactions in the absence of Ub (−Ub) were stopped at 30 min (b), 60 min (c, d), or 150 min (e). All reactions were analyzed by SDS-PAGE followed by Coomassie blue staining. Running positions of molecular mass markers (kDa), HDM2, UBA1 (E1), UbcH5b (E2), free Ub (Ub), and autoubiquitylated forms of HDM2 (*) are indicated. The experiments shown are representative of three independent experiments. Source data are provided as a Source Data file.