Journal: Molecules

Article Title: Characterization of Lysine Acetyltransferase Activity of Recombinant Human ARD1/NAA10

doi: 10.3390/molecules25030588

Figure Lengend Snippet: Figure 5. hARD1/NAA10-mediated lysine acetylation occurs under limited reaction conditions. (A) Autoacetylation activity of hARD1/NAA10 is dependent on the reaction time. A time-dependent in vitro acetylation assay was performed and western blot analysis was carried out to analyze the levels of acetylated protein. (B) hARD1/NAA10-mediated lysine acetylation in vitro is limited to a reaction time of 2 h. Hsp70 recombinant protein was incubated for different time points without or with purified rhARD1 protein. The acetylated levels were measured with immunoblotting. (C) Non-enzymatic acetylation reaction occurs at long incubation time. Autoacetylation of hARD1/NAA10 was analyzed with western blotting after 1 and 4 h incubation of hARD1/NAA10 WT and hARD1 DN mutant in in vitro acetylation assay. WT, wild-type; DN, dominant-negative mutant. (D) hARD1/NAA10-mediated lysine acetylation in vitro is favorable at pH 8.0. Hsp70 was subjected to in vitro acetylation assay in various pH with or without hARD1/NAA10. (E) hARD1/NAA10-mediated lysine acetylation of Hsp70 at pH 8.0. (F) Limited ratio of hARD1/NAA10 and Hsp70 to distinguish the enzymatic activity and the non-specific chemical reaction. The in vitro acetylation assays were performed in the varied ratio between hARD1/NAA10 and Hsp70.

Article Snippet: Anti-acetylated lysine (Lys-Ac) antibody (#9941) were purchased from Cell Signaling Technology.

Techniques: Activity Assay, In Vitro, Acetylation Assay, Western Blot, Recombinant, Incubation, Mutagenesis, Dominant Negative Mutation