Figure 2.

In vivo and in vitro ubiquitylation of RNF122. (A) HEK293T cells were seeded into 60-mm plates and transfected with 5 μg of Ub-HA and 5 μg of pcDB (empty) or wild-type or mutant RNF122-myc expression plasmid. At 48 h post-transfection, cell lysates in RIPA buffer were immunoprecipitated with anti-myc antibody agarose beads. The immunoprecipitates were analyzed on an immunoblot for HA. A smeared band was observed in the RNF122-myc group. (B) E2-selective ubiquitin ligase activity of RNF122. Bacterially expressed GST-RNF122ΔTM was incubated with ubiquitin, E1, and various E2 enzymes. Autoubiquitylation of RNF122 was detected by HRP-streptavidin immunoblotting. (C) RNF122 E3 ligase activity required an intact RING finger domain. The reactions were performed using ubiquitin, E1, E2 (UbcH5b or UbcH5c), and E3 (GST-RNF122ΔTM, or mutant GST-RNF122ΔTM), as indicated.