In vitro acetylation and ubiquitination assay

For vitro acetylation assay, purified His-NEDD4 (5 μg) was incubated with purified GST-P300-HAT (1 μg), purified GST-PCAF (1 μg) or P300-HA and P300-WY-HA immunoprecipitated from cell lysates with adding acetyl-coenzyme A (20 μM) and 5 x HAT assay buffer [250 mM Tris–HCl pH 8.0, 50% (v/v) glycerol, 0.5 mM EDTA, 5 mM dithiothreitol] in a total volume of 50 μL. The mixture was mixed mildly and placed for 2 h at 30°C. Then the contents were boiled in 1x SDS-loading buffer for immunoblotting with anti-acetyl-lysine antibody to detect NEDD4 acetylation.

For vitro ubiquitination assay, purified GST-VP40 was incubated with Myc-NEDD4, Myc-NEDD4_K535Q and Myc-NEDD4_K667Q immunoprecipitated from cell lysates in the presence of purified ubiquitin, E1, E2, Mg²⁺-ATP and DTT (50 mM) in a total reaction volume of 50 μL. The contents were mixed gently and incubated at 37°C for 1 h. Quench assays by addition of 50μL 2x SDS loading buffer followed by heating to 95°C for 5 minutes or 70°C for 10 minutes. The ubiquitination of VP40 was analyzed by immunoblotting with anti-GST antibody.