E1 ubiquitin activating enzyme ube1

FLAG-TRIM33 or its CA mutant was captured from transfected HEK 293T cell lysates by anti-FLAG agarose. After a thorough wash, the agarose was divided into aliquots. Empty anti-FLAG agarose was used as control. The in vitro ubiquitination assay was performed by incubating TRIM33-bound, mutant TRIM33-bound, or control agarose at 37°C for 1 h with E1 ubiquitin-activating enzyme UBE1, E2-conjugating enzyme UbcH5c, HA-ubiquitin, and ATP (all from Boston Biochem) in the presence or absence of purified GST–β-catenin WT or mutant or active PKCδ. The supernatant was removed after the assay and the agarose was thoroughly washed and analysed by IB.

Flag-KDM4C was captured from Flag-KDM4C plasmid-transfected HEK293T cell lysates by anti-Flag agarose beads. Empty anti-Flag agarose beads were used as a control. The in vitro ubiquitination assay was performed by incubating KDM4C or control agarose beads at 37°C for 1 hour with E1 ubiquitin-activating enzyme UBE1, E2-conjugating enzyme UbcH5c, HA-ubiquitin, and adenosine triphosphate (all from Boston Biochem) in the presence or absence of recombinant β-TrCP protein (Creative Biomart, BTRC-2545M). Then the supernatant was removed, and the beads were thoroughly washed and boiled in 1× loading buffer, followed by Western blot analysis with the indicated antibodies.