Ubquitination state of Eps15 and ATPα.

<p><b>A)</b> Ubiquitin enrichment from cytoplasmic extracts with elevated or decreased levels of Dube3a. Genotypes are listed below. Western blots were performed on ubiquitin-enriched extracts using Eps15 (top) and ATPα antibodies (bottom). Only the ∼100 kDa form of Eps15 could be detected in the elutions from the ubiquitin enrichment column for all genotypes. No ∼150 kDa form could be detected. The ∼100 kDa form appears to be ubiquitinated even in the <i>Dube3a^15b</i> mutant fractions. Ubiquitinated ATPα could be detected in all fractions except for the <i>Dube3a^15b</i> lane, indicating that ATPα may be ubiquitinated in a Dube3a dependent manner. <b>B)</b><i>In vitro</i> ubiquitin assays. Purified Dube3a and ATPα proteins were mixed with E1 and E2 proteins <i>in vitro</i> in an attempt to ubiquitinate ATPα using the Dube3a E3 ubiquitin ligase. The (+) and (–) symbols above the Western blot indicate the presence or absence of a component in the ubiquitination reaction run in that lane. ATPα protein could be detected in all of the appropriate lanes (red) and a Poly-Ub smear could be seen in the fourth lane from the left (the reaction which contained all components). <b>C)</b> The green signal (poly-Ub antibody) was used to quantify the level of ubiquitination detected above background (LANE 1) and above any Dube3a auto-ubiquitination (LANE 2). A strong smear detected in LANE 4 was 143%+/−15% above the background levels and was significantly higher than any signal that comes from Dube3a auto-ubiquitination (LANE 2). The (*) symbols indicates a p-value of less than 0.05 for these results by one way ANOVA analysis.