Figure 2.

Angelman syndrome mutants strongly interact with the S5a proteasome subunit. HEK293 were transfected with plasmids expressing the wild-type UBE3A and the C833A, L502P, and E550L mutants. After 24 h the cells were harvested and extracts incubated with the S5a GST fusion protein. After extensive washing, bound UBE3A proteins were detected by Western blotting using the anti-UBE3A antibody and are compared with the amount of UBE3A proteins present in 10% of the inputs. The lower panel shows a low exposure of the binding assay and in the bottom panel, Ponceau stain of the nitrocellulose membranes shows the levels of GST proteins used in the pull downs, with the arrows indicating the position of the GST–S5a fusion protein