Pha13 confers self-ubiquitination E3 ligase and ubiquitin binding ability.

<p>In vitro ubiquitination analysis was performed on recombinant proteins of Pha13, A20 mutant (Pha13A20m), AN1 mutant (Pha13AN1m), A20/AN1 double mutant (Pha13A20mAN1m) with or without FLAG-tagged ubiquitin (FLAG-Ub), ubiquitin-activating enzyme (E1), or ubiquitin-conjugating enzyme (E2). Ubiquitinated proteins were analyzed with immunoblotting using anti-FLAG antibodies. Coomassie staining was used as a loading control. The ubiquitinated Pha13 (Pha13-2Ub), E1 conjugated with one Ub (E1-Ub), E2 conjugated with one or two Ub (E2-Ub, E2-2Ub), and free Ub are indicated with arrow.</p>