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Effect of phosphorylation of Pellino 1 on its E3 ligase activity in the presence of different E2-conjugating enzymes. Pellino 1 was phosphorylated by incubation for 30 min at 30 °C without (lanes 1–3) or with (lanes 4–6) IRAK4, and its E3 ligase activity then measured for the times indicated by using UbcH5a (Top), UbcH5b (Middle), or UbcH4 (Bottom) as the E2-conjugating enzyme (see Experimental Procedures). pUb, polyubiquitin.
