Figure 3.

UBE3A wild-type, C->A, L502P and E550L mutants affect the proteolytic activity of the proteasome. HEK293 cells were transfected with UBE3A, C833A, L502P or empty vector as a control (a, b and c) or with UBE3A, L502P, E550L or empty vector as a control (e). Twenty-four hours post transfection, cells were collected and homogenized with 0.5% NP40 in dH20, and the proteolytic activity of the proteasome was assessed using the Proteasome Activity Assay Kit and a fluorescent microplate reader in the absence (a and e) or presence (b) of a proteasomal inhibitor. The collated results from three independent experiments to measure UBE3A effects on catalytic activity of the proteasome are shown in c at 10-min time point intervals. Standard deviations are also shown. (d) Shows the western blot of the wild-type and mutant UBE3A expression levels in HEK293 cells used in the assay shown in a. Also shown is the γ-tubulin loading control