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. 2021 Feb 19;30(6):430–442. doi: 10.1093/hmg/ddab050

Figure 3.

Figure 3

AS-associated missense mutations in both the N-terminal region and HECT domain of UBE3A affect catalytic activity. (A and B) Auto-ubiquitination and target ubiquitination of a representative set of UBE3A mutants and quantification of all mutants (C and D). Escherichia coli cells expressing E1, UbcH5 (E2) and with (+) or without (−) ubiquitin were transfected with a plasmid expressing mouse HA-tagged mUBE3A-iso3 (wild-type or the indicated mutant) and V5-RING1B-I53S, a catalytically inactive RING1B mutant that is unable to ubiquitinate itself. Cell lysates were analyzed by SDS-PAGE and immunoblotting using HA antibody to visualize UBE3A (A) and V5 antibody to visualize RING1B (B). Slower migrating, ubiquitinated UBE3A and RING1B species are indicated by a bracket and the unmodified protein band by an asterisk. Quantification of UBE3A auto-ubiquitination (WT, n = 9; mutants, n = 3) (C) and RING1B ubiquitination (WT, n = 8; mutants, n = 3) (D). Values are shown as the mean percent ± SEM of WT UBE3A. Unpaired Student’s t-test (two-tailed). Significant effects are indicated as *P < 0.05, **P < 0.005 and ***P < 0.001. Dashed grey lines depict 95% confidence interval. See Material and Methods section for detailed quantification and statistical methods.